Crossover analytical systems and methods using an immunosensor and magnetic immunosensor

ABSTRACT

The present invention relates to systems and methods that utilize a combination of immunoassay and magnetic immunoassay techniques to detect an analyte within an extended range of specified concentrations. In particular, a method includes determining a first concentration of an analyte at a first immunosensor from a reaction of a signal agent with a first complex of signal antibodies, the analyte, and capture antibodies immobilized on a surface of the first immunosensor, determining a second concentration of the analyte at a second immunosensor from a reaction of the signal agent with a second complex of the signal antibodies, the analyte, and capture antibodies immobilized on magnetic beads that are localized on or near a surface of the second immunosensor via a magnetic field, determining a weighted average of the first concentration and the second concentration, and comparing the weighted average to a predetermined crossover concentration point or zone.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/432,293 filed on Dec. 9, 2016, the entirety of which is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to systems and methods of determininganalytes in point-of-care testing. In particular, the present inventionrelates to systems and methods that utilize a combination of immunoassayand magnetic immunoassay techniques to detect an analyte within anextended range of specified concentrations.

BACKGROUND OF THE INVENTION

Point-of-care (POC) sample analysis systems are generally based on oneor more re-usable test instruments (e.g., a reading apparatus) thatperform sample tests using a single-use disposable testing device, e.g.,a cartridge or strip that contains analytical elements, e.g., electrodesor optics for sensing analytes such as pH, oxygen and glucose. Thedisposable testing device can include fluidic elements (e.g., conduitsfor receiving and delivering the sample to sensing electrodes oroptics), calibrant elements (e.g., aqueous fluids for standardizing theelectrodes with a known concentration of analyte), and dyes with knownextinction coefficients for standardizing optics. The instrument orreading apparatus contains electrical circuitry and other components foroperating the electrodes or optics, making measurements, and performingcomputations. The instrument or reading apparatus also has the abilityto display results and communicate those results to laboratory andhospital information systems (LIS and HIS, respectively), for example,via a computer workstation or other data management system.Communication between the instrument or reading apparatus and aworkstation, and between the workstation and a LIS or HIS, can be via,for example, an infrared link, a wired connection, wirelesscommunication, or any other form of data communication that is capableof transmitting and receiving electrical information, or any combinationthereof. A notable point-of-care system (The i-STAT® System, AbbottPoint of Care Inc., Princeton, N.J.) is disclosed in U.S. Pat. No.5,096,669, which comprises a disposable device, operating in conjunctionwith a hand-held analyzer, for performing a variety of measurements onblood or other fluids.

One benefit of point-of-care sample testing systems is the eliminationof the time-consuming need to send a sample to a central laboratory fortesting. Point-of-care sample testing systems allow a nurse or doctor(user or operator), at the bedside of a patient, to obtain a reliablequantitative analytical result, sometimes comparable in quality to thatwhich would be obtained in a laboratory. In operation, the nurse selectsa testing device with the required panel of tests, draws a biologicalsample from the patient, dispenses it into the testing device,optionally seals the testing device, and inserts the testing device intothe instrument or reading apparatus. While the particular order in whichthe steps occur may vary between different point-of-care systems andproviders, the intent of providing rapid sample test results close tothe location of the patient remains. The instrument or reading apparatusthen performs a test cycle, i.e., all the other analytical stepsrequired to perform the tests. Such simplicity gives the doctor quickerinsight into a patient's physiological status and, by reducing theturnaround time for diagnosis or monitoring, enables a quicker decisionby the doctor on the appropriate treatment, thus enhancing thelikelihood of a successful patient outcome.

Cardiac marker testing such as troponin testing is one such diagnostictest that benefits from the quicker turnaround time provided via POCsample analysis systems. National and international cardiologyguidelines have recommended a one-hour turnaround time for reportingresults of cardiac markers such as troponin to emergency departmentpersonnel, measured from the time of blood collection to reporting. Theuse of POC sample analysis systems reduce the turnaround times forreporting results of cardiac markers from that of central laboratoryassays, but current POC sample analysis systems are not as precise orsensitive as central laboratory assays. In fact, the gap in precisionand sensitivity between central laboratory assays and POC sampleanalysis systems is growing as manufacturers of central laboratoryassays have or will release troponin assays that have a 99^(th)percentile cutoff of about 10 ng/L and a limit of detection of <1 ng/L,which is presently not possible for current POC testing assays. Thesehigh-sensitivity assays are able to detect troponin in the majority ofhealthy subjects, and clinically, this allows for the detection of morecases of myocardial injury.

In order to compete analytically with these central laboratory assays,next generation POC testing assays will need to make technologicadvancements. Thus there remains a need for systems and methods toextend the range of sensitivity for sample testing devices, e.g.,single-use blood testing cartridges, used with one or more testinstruments at the POC in a hospital or other location for deliveringmedical care.

SUMMARY OF THE INVENTION

In one embodiment, an immunoassay system is provided for determining aconcentration of an analyte in a sample over an extended concentrationrange. The system includes a first electrochemical sensor including animmobilized layer of antibodies configured to bind to a first complex ofsignal antibodies and the analyte to form a second complex, and a secondelectrochemical sensor having a magnetic field disposed locally aroundthe second electrochemical sensor. The magnetic field is configured toattract magnetic beads onto a surface of the second electrochemicalsensor such that a third complex of the first complex and antibodiesimmobilized on the magnetic beads is localized on the secondimmunosensor. The system further includes a fluid comprising asubstrate, one or more processors, and non-transitory machine readablestorage medium storing a set of instructions which when executed by theone or more processors cause the one or more processors to: measure afirst electrochemical signal at the first electrochemical sensor from areaction of the substrate with the second complex, measure a secondelectrochemical signal at the second electrochemical sensor from areaction of the substrate with the third complex, determine a firstconcentration of the analyte in the sample from the firstelectrochemical signal, determine a second concentration of the analytein the sample from the second electrochemical signal, determine aweighted average of the first concentration and the secondconcentration, compare the weighted average of the first concentrationand the second concentration to a predetermined crossover concentrationpoint. When the weighted average is above the crossover concentrationpoint, the first concentration is reported as a final concentration ofthe analyte in the sample from the first electrochemical signal. Whenthe weighted average is below the crossover concentration point, thesecond concentration is reported as the final concentration of theanalyte in the biological sample.

Optionally, system further includes a first reagent region coated withthe signal antibodies for the analyte, and a second reagent regioncoated with the magnetic beads. The first electrochemical sensor, secondelectrochemical sensor, the first reagent region, and the second reagentregion may be located on a sensor chip.

In some embodiments, the system further includes a composite materialincluding a binder such as polyimide or polyvinyl alcohol (PVA), and aparticulate magnetic material. The particulate magnetic material iscomprised of neodymium iron boron (NdFeB) alloy or aluminum nickelcobalt (AlNiCo) alloy, and configured to generate the magnetic field.

In another embodiment, an immunoassay system is provided for determininga concentration of an analyte in a sample over an extended concentrationrange. The system includes a first electrochemical sensor including animmobilized layer of antibodies configured to bind to a first complex ofsignal antibodies and the analyte to form a second complex, and a secondelectrochemical sensor having a magnetic field disposed locally aroundthe second electrochemical sensor. The magnetic field is configured toattract magnetic beads onto a surface of the second electrochemicalsensor such that a third complex of the first complex and antibodiesimmobilized on the magnetic beads is localized on the secondimmunosensor. The system further includes a fluid comprising asubstrate, one or more processors, and non-transitory machine readablestorage medium storing a set of instructions which when executed by theone or more processors cause the one or more processors to: measure afirst electrochemical signal at the first electrochemical sensor from areaction of the substrate with the second complex, measure a secondelectrochemical signal at the second electrochemical sensor from areaction of the substrate with the third complex, determine a firstconcentration of the analyte in the sample from the firstelectrochemical signal, determine a second concentration of the analytein the sample from the second electrochemical signal, determine aweighted average of the first concentration and the secondconcentration, and compare the weighted average of the firstconcentration and the second concentration to a predetermined crossoverconcentration zone. When the weighted average is above the crossoverconcentration zone, the first concentration is reported as a finalconcentration of the analyte in the sample from the firstelectrochemical signal. When the weighted average is below the crossoverconcentration zone, the second concentration is reported as the finalconcentration of the analyte in the biological sample from the secondelectrochemical signal. When the weighted average is within thecrossover concentration zone, the weighted average is reported as thefinal concentration of the analyte in the biological sample.

Optionally, system further includes a first reagent region coated withthe signal antibodies for the analyte, and a second reagent regioncoated with the magnetic beads. The first electrochemical sensor, secondelectrochemical sensor, the first reagent region, and the second reagentregion may be located on a sensor chip.

In some embodiments, the system further includes a composite materialincluding a binder such as polyimide or polyvinyl alcohol (PVA), and aparticulate magnetic material. The particulate magnetic material iscomprised of neodymium iron boron (NdFeB) alloy or aluminum nickelcobalt (AlNiCo) alloy, and configured to generate the magnetic field.

In another embodiment, non-transitory machine readable storage medium isprovided for having instructions stored thereon that when executed byone or more processors cause the one or more processors to perform amethod comprising measuring a first signal at a first immunosensor froma reaction of a signal agent with a first complex of signal antibodies,analyte, and capture antibodies immobilized on a surface of the firstimmunosensor, measuring a second signal at a second immunosensor from areaction of the signal agent with a second complex of the signalantibodies, the analyte, and capture antibodies immobilized on magneticbeads that are localized on or near a surface of the second immunosensorvia a magnetic field, determining a first concentration of the analytein the sample from the first electrochemical signal, determining asecond concentration of the analyte in the sample from the secondelectrochemical signal, determining a weighted average of the firstconcentration and the second concentration, and comparing the weightedaverage of the first concentration and the second concentration to apredetermined crossover concentration point. When the weighted averageis greater than the crossover concentration point, the firstconcentration is reported as a final concentration of the analyte in thesample from the first signal. When the weighted average is less than thecrossover concentration point, the second concentration is reported asthe final concentration of the analyte in the biological sample from thesecond signal.

In yet another embodiment, non-transitory machine readable storagemedium is provided for having instructions stored thereon that whenexecuted by one or more processors cause the one or more processors toperform a method comprising measuring a first signal at a firstimmunosensor from a reaction of a signal agent with a first complex ofsignal antibodies, analyte, and capture antibodies immobilized on asurface of the first immunosensor, measuring a second signal at a secondimmunosensor from a reaction of the signal agent with a second complexof the signal antibodies, the analyte, and capture antibodiesimmobilized on magnetic beads that are localized on or near a surface ofthe second immunosensor via a magnetic field, determining a firstconcentration of the analyte in the sample from the firstelectrochemical signal, determining a second concentration of theanalyte in the sample from the second electrochemical signal,determining a weighted average of the first concentration and the secondconcentration, and comparing the weighted average of the firstconcentration and the second concentration to a predetermined crossoverconcentration zone. When the weighted average is greater than thecrossover concentration zone, the first concentration is reported as afinal concentration of the analyte in the sample from the first signal.When the weighted average is less than the crossover concentration zone,the second concentration is reported as the final concentration of theanalyte in the biological sample from the second signal. When theweighted average is within the crossover concentration zone, theweighted average is reported as the final concentration of the analytein the biological sample from the first signal and the second signal.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood in view of the followingnon-limiting figures, in which:

FIG. 1 illustrates the evolution of Troponin immunoassays in accordancewith some aspects of the invention;

FIG. 2 illustrates the principle of a combined immunoassay accordancewith some aspects of the invention;

FIG. 3 shows a point-of-care instrument system in accordance with someaspects of the invention;

FIGS. 4 and 5A-5J show sensing devices or cartridges in accordance withsome aspects of the invention;

FIG. 6A shows a side view of the fabrication of a sensor chip inaccordance with some aspects of the invention;

FIGS. 6B, 7, 8A, and 8B show sensor chip configurations in accordancewith some aspects of the invention;

FIGS. 9A-9C illustrate various exemplary configurations for thepositioning of a magnet below a sensor chip within a cartridge inaccordance with some aspects of the invention;

FIG. 10 illustrates an exemplary configuration for the positioning ofsensors on a sensor chip within a cartridge in accordance with someaspects of the invention;

FIGS. 11A and 11B show exemplary immunosensors partially covered with aprinted magnetic layer leaving a portion of the perimeter of theimmunosensor exposed in accordance with some aspects of the invention;

FIG. 12 illustrates an etched trench process in accordance with someaspects of the invention;

FIG. 13 illustrates an exemplary configuration for the positioning ofsensors on a sensor chip within a cartridge in accordance with someaspects of the invention;

FIGS. 14-17 show processes in accordance with some aspects of theinvention; and

FIG. 18 shows a graph that illustrates the impact of being able todetermine a concentration of an analyte in a sample over an extendedconcentration range in accordance with some aspects of the invention.

DETAILED DESCRIPTION OF THE INVENTION Introduction

Cardiac troponin (cTn) is the primary biomarker used in the diagnosis ofacute myocardial infarction (AMI) and risk stratification for futureadverse cardiac events. However, the analytical sensitivity gap betweencentral laboratory assays and POC sample analysis systems for cardiactroponin testing has grown and can hinder the adoption of POC testingfor some hospitals. There may also be a need for POC sample analysissystems that can detect other biomarkers or multiple biomarkers. Forexample, while cardiac troponin is the primary analyte for AMIdiagnosis, B-type natriuretic peptide (BNP) and NT-proBNP have shown tobe useful for short-term risk stratification. The detection of highsensitivity cardiac troponin (hs-cTn) might also be useful as a riskstratification marker in primary care, i.e., for patients who areasymptomatic. This is based on observations that increased cardiactroponin is associated with a high risk for adverse cardiac outcomes inthe absence of acute coronary syndromes. If detection of thesebiomarkers becomes adopted as part of routine medical care for high riskpatients, then POC testing for hs-cTn may be useful and convenient whentested in physician offices and clinics.

Troponins are generally undetectable in healthy patients. The absoluteabnormal value for troponins varies depending on the clinical setting inwhich the patient is evaluated and the assay used. In a patient whopresents with chest pain and possible myocardial infarction (MI), anabnormal value is typically above the 99^(th) percentile of the healthypopulation as a cutoff using an assay with acceptable precision. The99^(th) percentiles for cTnT and cTnI detection are well known as 0.012to 0.016 ng/mL and 0.008 to 0.058 ng/mL, respectively. The wider rangeof the 99^(th) percentile concentrations for the cTnI assay stems fromthe many different detection assays using different antibodies and assayapproaches. POC cTn assays often have higher 99^(th) percentile valuesdue in part to increased analytical noise and lower sensitivity ascompared to the current laboratory cTn assays. For example, the 99^(th)percentile cutoff point for cTnT detection in central laboratory assaysis well-known at 0.01 ng/mL. In contrast, the 99th percentile cutoffpoint for cTnT detection in troponin POC sample analysis systems istypically around 0.05 to 0.08 ng/mL.

Troponin POC sample analysis systems are typically based on the reactionof the analyte with antibodies. Within the finite limits of thedetection zone, the analytical sensitivity is a direct function of theability of the assay to capture as much of the analyte as possible withoptimal precision. Optimal precision, as described by the coefficient ofvariation (CV) at the 99^(th) percentile of the upper reference limitfor each assay (as shown in FIG. 1), is generally defined as less thanor equal to ten percent. Better precision (CV of less than or equal toten percent) allows for more sensitive assays and facilitates thedetection of changing values and lowers the 99^(th) percentile decisionlimits of the assay. Nonetheless, developing POC sample analysis systemsthat meet these needs and lowers the 99^(th) percentile decision limitsof the assay has been challenging.

Enhancement of assay performance requires increasing the resolutionbetween (i) the limit of blank (LoB) and the limit of detection (LoD)and (ii) the LoD and the 99^(th) percentile. LoB is the highest apparentanalyte concentration expected to be found when replicates of a blanksample containing no analyte are tested. LoD is the lowest analyteconcentration likely to be reliably distinguished from the LoB and atwhich detection is feasible. LoD is determined by utilizing both themeasured LoB and test replicates of a sample known to contain a lowconcentration of analyte. Limit of Quantitation (LoQ) is the lowestconcentration at which the analyte can not only be reliably detected butat which some predefined goals for bias and imprecision are met. The LoQmay be equivalent to the LoD or it could be at a much higherconcentration. Sensitivity, analytical sensitivity, lower limit ofdetection, LoB, LoD, and LoQ are all terms used to describe the smallestconcentration of an analyte that can be reliably measured by the assay.

One of the ways to improve the sensitivity or increase the resolutionbetween (i) the LoB and the LoD and (ii) the LoD and the 99^(th)percentile in an immunoassay is to improve the signal to noise ratio.For example, improvement to sensitivity in an immunoassay may beachieved by increasing the signal generating ability of the system ordecreasing the background signal generated by the system. The signalgenerating ability may be considered in terms of the “sensitivity slope”or the amount of signal generated per unit of analyte: slope=(Current(nA))/(Concentration (ng/ml)), and thus the Concentration(ng/ml)=(Current (nA))/(slope (nA/ng/ml). In conventional POC sampleanalysis systems such as those described in U.S. Pat. No. 7,419,821,which is incorporate herein by reference in its entirety, a sensor iscoated with a biolayer comprising a covalently attached anti-troponinantibody, to which a complex of troponin and enzyme-antibody conjugatebinds. The enzyme-antibody conjugate is thereby immobilized close to theelectrode in proportion to the amount of troponin initially present inthe sample. In addition to specific binding, the enzyme-antibodyconjugate may bind non-specifically to the sensor. Non-specific bindingprovides a background signal from the sensor that is undesirable andshould be minimized. To solve this problem, U.S. Pat. No. 7,419,821discloses the use of rinsing protocols, and in particular the use ofsegmented fluid to rinse the sensor, as a means to decrease thebackground signal. POC sample analysis systems such as those describedin U.S. Pat. No. 7,419,821 have a signal generating ability or“sensitivity slope” of about 4 nA/ng/ml and are particularly effectivefor the detection of high levels of a biomarker such as troponin (i.e.,high-end sensitivity).

However, based on a sample size of 10 μL, which is typically of POCsample analysis systems, and a number of analyte molecules that may bepresent in such a sample size, the theoretical maximum slope is about1200 nA/ng/mL. It is believed that the conventional POC sample analysissystems merely have a signal generating ability of about 4 nA/ng/mlbecause the biolayer comprising the covalently attached anti-troponinantibody is immobilized on, or close to, the sensor surface, and thusonly analyte brought into contact with the sensor surface is subject tocapture and analysis (e.g., an estimated 0.3% of all analyte in thesample is subject to capture and analysis).

In order to increase the signal generating ability or “sensitivityslope” beyond 4 nA/ng/ml and increase the effectiveness of animmunoassay for the detection of low levels of a biomarker such astroponin (i.e., low-end sensitivity), conventional POC sample analysissystems such as those described in U.S. Pat. No. 9,233,370, which isincorporate herein in its entirety, were developed with magneticallysusceptible bead capture techniques. The magnetically susceptible beadcapture techniques allow for the enzyme-antibody conjugate to belocalized on, or close to, the sensor surface and function tosubstantially retain the enzyme-antibody conjugate at or near the sensorduring removal of the unbound sample and washing of the sensor to removethe non-specific binding. POC sample analysis systems such as thosedescribed in U.S. Pat. No. 9,233,370 have a signal generating ability or“sensitivity slope” of about 40 nA/ng/ml (i.e., 10× the signalgenerating ability of non-magnetic immunoassays) and are particularlyeffective for the detection of low levels of a biomarker such astroponin (i.e., low-end sensitivity). Nonetheless, conventional POCsample analysis systems are far from achieving the theoretical maximumslope of about 1200 nA/ng/mL.

In order to improve upon the signal generating ability of conventionalPOC sample analysis systems and increase the effectiveness of animmunoassay for the detection of low levels of a biomarker such astroponin (i.e., low-end sensitivity), one embodiment of the presentinvention is directed to an extended range magnetic sensor device havinga fixed antibody capture site situated over a first sensor (e.g., anamperometric sensor) and another antibody capture site situated over asecond sensor (e.g., an amperometric sensor) with a high field magnetpositioned underneath. The two sensors each have sensitivity to ananalyte (e.g., cTn) but with different sensitivities due to thedifference in the capture reagents being used for each respectivesensor. The first sensor is typically the lower sensitivity sensor (aslope of less than 5 nA/ng/ml) and is particularly effective for thedetection of high levels of an analyte such as troponin (i.e., high-endsensitivity). The second sensor is typically the higher sensitivitysensor (a slope of greater than 7 nA/ng/ml) and is particularlyeffective for the detection of low levels of an analyte such as troponin(i.e., low-end sensitivity). Consequently, the implementation of boththe lower sensitivity sensor and the high sensitivity sensor on a singledevice extends the range of concentrations of an analyte that may bedetected using the device.

The difference in the location of the analyte and label reagent bindingbetween the two sensors accounts largely for their difference insensitivities to the analyte. The sensitivity differences between thetwo sensors may be further controlled by variation of the time betweenthe dissolution of the paramagnetic reagent into the sample and thesample's positioning over the first sensor. Further control of thesensitivities between the two sensors may be achieved by altering theconcentration of the paramagnetic antibody coated particles used in theassay. Another technique of controlling the sensor sensitivities may bethrough control of antibody concentration, affinities or avidities onthe first sensor and the paramagnetic reagents.

The advantage of the aforementioned technical solution for improvingupon the signal generating ability of POC sample analysis systems andincreasing the effectiveness of an immunoassay for the detection of lowlevels of a biomarker such as troponin (i.e., low-end sensitivity) isthat it will eliminate the technical problems with increasing theresolution between (i) the limit of blank (LoB) and the limit ofdetection (LoD) and (ii) the LoD and the 99^(th) percentile. Forexample, implementations of the present invention provide a technicalcontribution over conventional POC sample analysis systems and methodsbecause the technical features of the present invention interoperate toprovide both the lower sensitivity sensor and the high sensitivitysensor on a single device, which extends the range of concentrations ofan analyte that may be detected using the device.

Immunoassays

FIG. 2 illustrates the principle of a combined immunoassay (e.g., aone-step combined immunoassay) 200 according to specific embodiments ofthe present invention that extends the range of concentrations of atarget analyte such as troponin I (TnI) or cardiac troponin I (cTnI),which may be detected using an analyzer. In various embodiments, thecombined immunoassay 200 includes a non-magnetic immunoassay technique205 that utilizes an enzyme-biomolecule conjugate 210 configured to bindto the target analyte 215 and a capture biomolecule 220 (e.g., latexbeads or microspheres coated with capture biomolecule) immobilized on ornear a surface of a non-magnetic sensor (i.e., a heterogeneous surfacecapture immunosensor). The capture biomolecule 220 is configured to bindto the target analyte 215 that is bound to the enzyme-biomoleculeconjugate 210 such that the enzyme-biomolecule conjugate 210 is capturedand immobilized on or near a surface of the non-magnetic sensor. Thenon-magnetic sensor may be either clamped at a fixed electrochemicalpotential sufficient to oxidize or reduce a product of a hydrolyzedsubstrate but not the substrate directly, or the potential may be sweptone or more times through an appropriate range. The combined immunoassay200 further includes a magnetic immunoassay technique 225 that utilizesthe enzyme-biomolecule conjugate 210 configured to bind to the targetanalyte 215 and a capture biomolecule 230 (e.g., magnetic beads ormicrospheres coated with capture biomolecule). The capture biomolecule230 is configured to bind to the target analyte 215 that is bound to theenzyme-biomolecule conjugate 210. The capture biomolecule 230 bound tothe target analyte 215 that is bound to the enzyme-biomolecule conjugate210 may be attracted via a magnet onto or near a surface of a magneticsensor (i.e., a homogeneous magnetic bead capture immunosensor) suchthat the enzyme-biomolecule conjugate 210 is captured and immobilized onor near a surface of the magnetic sensor. The magnetic sensor may beeither clamped at a fixed electrochemical potential sufficient tooxidize or reduce a product of a hydrolyzed substrate but not thesubstrate directly, or the potential may be swept one or more timesthrough an appropriate range.

The enzyme-biomolecule conjugate 210 includes an enzyme conjugated tobiomolecules selected to bind to an analyte of interest. In someembodiments, the enzyme is alkaline phosphatase (ALP), horseradishperoxidase, or glucose oxidase and the biomolecules are chosen fromamong ionophores, cofactors, polypeptides, proteins, glycopeptides,enzymes, immunoglobulins, antibodies, antigens, lectins, neurochemicalreceptors, oligonucleotides, polynucleotides, DNA, RNA, or suitablemixtures. In some embodiments, the biomolecules may be selected to bindto one or more of human chorionic gonadotrophin, troponin I, troponin T,troponin C, a troponin complex, creatine kinase, creatine kinase subunitM, creatine kinase subunit B, myoglobin, myosin light chain, or modifiedfragments of these. Such modified fragments are generated by oxidation,reduction, deletion, addition or modification of at least one aminoacid, including chemical modification with a natural moiety or with asynthetic moiety. For example, the biomolecules may be selected as amonoclonal or polyclonal anti-troponin I antibody (e.g.,BiosPacific—Peptide 4 (G-130-C), HyTest—560 (19C7, Cat#4T21—monoclonalTroponin I Ab) and International Point of Care—8I7 (Cat# MA-1040). Incertain embodiments, the biomolecule binds to the analyte specificallyand has an affinity constant for binding analyte ligand of about 10⁷ to10¹⁵ M⁻¹.

The capture biomolecule 220 may be provided as a biolayer deposited ontoor near at least a portion of the non-magnetic sensor. A biolayer is aporous layer comprising on its surface a sufficient amount ofbiomolecules that can either bind to an analyte of interest, or respondto the presence of such analyte by producing a change that is capable ofmeasurement. Optionally, a permselective screening layer may beinterposed between the non-magnetic sensor and the biolayer to screenelectrochemical interferents as described in U.S. Pat. No. 5,200,051,which is incorporated herein in its entirety.

In some embodiments, the biolayer is constructed from latex beads ofspecific diameter in the range of about 0.001 to 50 microns (e.g.,ThermoFisher OptiLink Carboxylate-Modifies Microparticles(Catalog#83000591100351), 0.2 um diameter). The beads may be modified bycovalent attachment of any suitable biomolecules that can either bind toan analyte of interest, or respond to the presence of such analyte byproducing a change that is capable of measurement. Many methods ofattachment exist in the art, including providing amine reactiveN-hydroxysuccinimide ester groups for the facile coupling of lysine orN-terminal amine groups of proteins. In certain embodiments, thebiomolecules are chosen from among ionophores, cofactors, polypeptides,proteins, glycopeptides, enzymes, immunoglobulins, antibodies, antigens,lectins, neurochemical receptors, oligonucleotides, polynucleotides,DNA, RNA, or suitable mixtures. In some embodiments, the biomoleculesmay be selected to bind one or more of human chorionic gonadotrophin,troponin I, troponin T, troponin C, a troponin complex, creatine kinase,creatine kinase subunit M, creatine kinase subunit B, myoglobin, myosinlight chain, or modified fragments of these. Such modified fragments aregenerated by oxidation, reduction, deletion, addition or modification ofat least one amino acid, including chemical modification with a naturalmoiety or with a synthetic moiety. For example, the biomolecules may beselected as a monoclonal or polyclonal anti-troponin I antibody (e.g.,SDIX—M06 (# D2440MA06-MA) and HyTest—Cap1 (19C7, Cat#4T21—monoclonalTroponin I Ab). In certain embodiments, the biomolecule binds to theanalyte specifically and has an affinity constant for binding analyteligand of about 10⁷ to 10¹⁵ M⁻¹¹.

The capture biomolecule 230 may be provided as biomolecules attached tomagnetically susceptible beads. The magnetically susceptible beads maybe comprised of any material known in the art that is susceptive tomovement by a magnet (e.g., permanent magnet or electromagnet) utilizedin or in concert with the device of the present invention. As such, theterms “magnetic” and “magnetically susceptible” with regard to beads canbe used interchangeably.

In some embodiments, the beads include a magnetic core, which preferablyis completely or partially coated with a coating material. The magneticcore may comprise a ferromagnetic, paramagnetic or a superparamagneticmaterial. In preferred embodiments, the magnetically susceptible beadscomprise a core and an outer polymer coating. In other embodiments, themagnetic beads comprise non-magnetic substrate beads formed, forexample, of a material selected from the group consisting ofpolystyrene, polyacrylic acid and dextran, upon which a magnetic coatingis placed. In certain embodiments where the magnetically susceptiblebeads comprise a core, the magnetic core may comprise one or more offerrite, Fe, Co, Mn, Ni, metals comprising one or more of theseelements, ordered alloys of these elements, crystals comprised of theseelements, magnetic oxide structures, such as ferrites, and combinationsthereof. In other embodiments where the magnetically susceptible beadscomprise a core, the magnetic core may be comprised of magnetite(Fe₃O₄), maghemite (γ-Fe₂O₃), or divalent metal-ferrites provided by theformula Me_(1-x)OFe_(3+x)O₃ where Me is, for example, Cu, Fe, Ni, Co,Mn, Mg, or Zn or combinations of these materials, and where x rangesfrom 0.01 to 99. Suitable materials for the outer polymer coating overthe core include synthetic and biological polymers, copolymers andpolymer blends, and inorganic materials. Polymer materials may includevarious combinations of polymers of acrylates, siloxanes, styrenes,acetates, akylene glycols, alkylenes, alkylene oxides, parylenes, lacticacid, and glycolic acid. Biopolymer materials include starch or similarcarbohydrate. Inorganic coating materials may include any combination ofa metal, a metal alloy, and a ceramic. Examples of ceramic materials mayinclude hydroxyapatite, silicon carbide, carboxylate, sulfonate,phosphate, ferrite, phosphonate, and oxides of Group IV elements of thePeriodic Table of Elements.

In principal, any correctly-sized magnetically susceptible bead capableof being positioned with the magnet of the present invention may beutilized, taking into account the dispersability requirements for themagnetically susceptible beads. In preferred embodiments, at least 50wt. %, e.g., at least 75 wt. %, of the magnetically susceptible beadsare retained at or near the sensor surface. In some exemplaryembodiments, the average particle size of the magnetically susceptiblebeads may range from 0.01 μm to 20 μm, e.g., from 0.1 μm to 10 μm, from0.1 μm to 5 μm or from 0.2 μm to 1.5 μm. As used herein, the term“average particle size” refers to the average longest dimension of theparticles, e.g., beads, for example the diameter for sphericalparticles, as determined by methods well-known in the art. The particlesize distribution of the magnetically susceptible beads preferably isunimodal, although polymodal distributions may also be used inaccordance with the present invention. While use of a sphericalmagnetically susceptible bead is preferred, in other embodiments, otherbead shapes and structures, e.g., ovals, sub-spherical, cylindrical andother irregular shaped particles, are within the meaning of the term“beads” and “microparticles” as used herein.

Commercial sources for magnetically susceptible bead preparationsinclude Invitrogen™ (Carlsbad, Calif., U.S.A.) by Life Technologies™,Ademtech (Pessac, France), Chemicell GmbH (Berlin, Germany), BangsLaboratories, Inc.™ (Fishers, Ind.) and Seradyn, Inc. (Indianapolis,Ind.) (e.g., Invitrogen™ by Life™ Technologies—Dynabeads® MyOne™Streptavidin T1 (Catalog#65601/65602), 1 um diameter). Many of thecommercially available products incorporate surface functionalizationthat can be employed to immobilize biomolecules such as antibodies(e.g., IgG) on the bead surfaces. Exemplary functionalizations includecarboxyl, amino or streptavidin-modified magnetically susceptible beads.

In some embodiments, the magnetically susceptible beads are coated withany suitable biomolecules that can either bind to an analyte ofinterest, or respond to the presence of such analyte by producing achange that is capable of measurement. Many methods of attachment existin the art, including providing amine reactive N-hydroxysuccinimideester groups for the facile coupling of lysine or N-terminal aminegroups of proteins. In the instance of streptavidin-modifiedmagnetically susceptible beads, the biomolecules may be modified toinclude a binder such as biotin to attach the biomolecules on the beadsurfaces. For example, the biomolecules may be attached to biotin (e.g.,Thermo Scientific—EZ-link Sulfo-NHS-LC-LC-biotin (Product#21338) orEZ-link Sulfo-NHS-LC-biotin (Product#21335)). In certain embodiments,the biomolecules are chosen from among ionophores, cofactors,polypeptides, proteins, glycopeptides, enzymes, immunoglobulins,antibodies, antigens, lectins, neurochemical receptors,oligonucleotides, polynucleotides, DNA, RNA, or suitable mixtures. Thebiomolecules may be selected to bind one or more of human chorionicgonadotrophin, troponin I, troponin T, troponin C, a troponin complex,creatine kinase, creatine kinase subunit M, creatine kinase subunit B,myoglobin, myosin light chain, or modified fragments of these. Suchmodified fragments are generated by oxidation, reduction, deletion,addition or modification of at least one amino acid, including chemicalmodification with a natural moiety or with a synthetic moiety. Forexample, the biomolecules may be selected as a monoclonal or polyclonalanti-troponin I antibody (e.g., BiosPacific—Peptide 3 (G-129-C) andHyTest—Cap1 (19C7, Cat#4T21—monoclonal Troponin I Ab). In certainembodiments, the biomolecule binds to the analyte specifically and hasan affinity constant for binding analyte ligand of about 10⁷ to 10¹⁵M⁻¹¹.

As should be understood, embodiments of the present invention may beimplemented in a variety of different systems and contexts. Certainembodiments are particularly applicable to immunoassays that detect anenzymatically produced electroactive species (e.g., 4-aminophenol) fromthe reaction of a substrate (e.g., 4-aminophenylphosphate) with theantibody-enzyme conjugate (e.g., one or more antibodies bound toalkaline phosphatase (ALP). However, the systems and techniquesdescribed herein may be used to detect an analyte using biomoleculesother than antibodies labeled with various labels beyond enzymes. Forexample, the biomolecules described herein may be attached to labelsincluding a radiolabel, chromophore, flurophore, chemiluminescentspecies, ionophore, electroactive species and others known in the artwithout departing from the spirit and scope of the present invention.

As should be further understood, embodiments of the present inventionmay be implemented in a variety of different systems and configurations,and the term on or near a surface of the sensor is used herein todescribe the relationship between a biomolecule complex and the surfaceof a particular sensor. On or near a surface of a sensor defines aworking distance between the biomolecule complex and the surface of theparticular sensor that needs to be maintained such that a signalgenerated by a reaction of the biomolecule complex with a substrate canbe measured at the surface of the particular sensor. In someembodiments, the working distance is less than 800 μm, for example lessthan 600 μm or less than 500 μm.

Biological Sample Test System for Performing Immunoassays

The present invention relates to a handheld POC instrument systemincluding a self-contained disposable sensing device or cartridge(device(s)) and a reader or analyzer (instrument(s)) configured for useat a patient bedside. A fluid sample to be measured is drawn into asample entry orifice or port in the cartridge and the cartridge isinserted into the analyzer through a slotted opening or port.Measurements performed by the analyzer are output to a display or otheroutput device, such as a printer or data management system via a port onthe analyzer to a computer port. Transmission can be via Wi-Fi,Bluetooth link, infrared and the like. For example, the handheld IVDinstrument system may be of similar design to the systems disclosed inU.S. Pat. No. 5,096,669 and U.S. Pat. No. 7,419,821, both of which areincorporated herein by reference in their entireties.

FIG. 3 shows the component parts and interactions of a typical handheldPOC instrument system. The system 300 may include an analyzer 305, adisposable sensing device 310, and a central data station or datamanager 315. The analyzer 305 may include, for example, a display 320for visual reference and one or more input devices 325 for data entry.The one or more input devices 325 may include one or more mechanismsthat permit an operator to input information to analyzer 305, such as,but not limited to, a touch pad, dial, click wheel, scroll wheel, touchscreen, one or more buttons (e.g., a keyboard), mouse, game controller,track ball, microphone, camera, proximity sensor, light detector, motionsensors, biometric sensor, and combinations thereof. The sensing device310 may include, for example, a port 330 for receiving a patient sampleand a sensor array 335 for detecting an analyte in a biological sample.For example, the sensing device 310 may be configured to performanalyses on a range of biological sample types. These sample types mayinclude, for example, blood, plasma, serum, sputum, cerebrospinal fluid,tears, urine, body tissue, fecal matter, and the like. The sensingdevice 310 may be inserted into the analyzer 305 through an opening 340such that the analyzer 305 is in electrical contact with the sensingdevice 310 for implementing the functionality, steps, and/or performanceof the present invention.

The analyzer 305 may communicate with the data manager 315 using, forexample, a wireless connection, an infrared link, an optical link, anetwork connection 345, 350, or any other form of communication linkthat uses any form of communication protocol to transfer information.The data manager 315 can be resident on a network infrastructure such aswithin a cloud environment, or may be a separate independent computingdevice (e.g., a computing device of a service provider). The datamanager 315 may include a bus, processor, a storage device, a systemmemory (hardware device), one or more input devices, one or more outputdevices, and a communication interface. The data manager 315 may beconfigured to provide connectivity between the analyzer 305 and centrallocations, such as, for example, a LIS or HIS (laboratory or hospitalinformation system), and sensing device 305. The data manager 315 may beconnected with the various system constituents using any type ofcommunications connection that is capable of transmitting and receivingelectronic information, such as, for example, an Ethernet connection orother computer network connection. The data manager 315 can alsooptionally provide a direct link back to a vendor's (productmanufacturer) information system, for example via the Internet, adial-up connection or other direct or indirect communication link, orthrough the LIS or HIS. Such an exemplary embodiment can provide forautomated re-ordering of sensing devices 305 to maintain predeterminedlevels of inventory at a hospital and allow the vendor to forecastdemand and adequately plan the manufacture of the devices 305. It canalso provide a means for updating device information, e.g. cartridgeattributes and profiles, and control fluid information, e.g. expectedanalyte test ranges.

The analyzer 305 may further include a processor, a storage device, andsystem memory. The processor may be one or more conventional processors,microprocessors, or specialized dedicated processors that includeprocessing circuitry operative to interpret and execute computerreadable program instructions, such as program instructions forcontrolling the operation and performance of one or more of the variousother components of the analyzer 305 and/or sensing device 310 forimplementing the functionality, steps, and/or performance of the presentinvention. In certain embodiments, the processor interprets and executesthe processes, steps, functions, and/or operations of the presentinvention, which may be operatively implemented by the computer readableprogram instructions. For example, the processor can measure a signalgenerated at a sensor of the sensing device 310 (e.g., a signalindicative of the presence and/or concentration of an analyte in abiological sample), determine a concentration of the analyte in thebiological sample based on the measured signal, and report thedetermined concentration (e.g., display the determined concentration ondisplay 320). In some embodiments, the information obtained or generatedby the processor, e.g., the identity of the sensing device 310, theshelf-life of the sensing device 310, the determined concentration,etc., can be stored in the storage device.

The storage device may include removable/non-removable,volatile/non-volatile computer readable media, such as, but not limitedto, non-transitory machine readable storage medium such as magneticand/or optical recording media and their corresponding drives. Thedrives and their associated computer readable media provide for storageof computer readable program instructions, data structures, programmodules and other data for operation of analyzer 305 in accordance withthe different aspects of the present invention. In embodiments, storagedevice may store an operating system, application programs, and programdata in accordance with aspects of the present invention.

The system memory may include one or more storage mediums, including forexample, non-transitory machine readable storage medium such as flashmemory, permanent memory such as read-only memory (“ROM”),semi-permanent memory such as random access memory (“RAM”), any othersuitable type of non-transitory storage component, or any combinationthereof. In some embodiments, an input/output system (BIOS) includingthe basic routines that help to transfer information between the variousother components of the analyzer 305 and system 300, such as duringstart-up, may be stored in the ROM. Additionally, data and/or programmodules, such as at least a portion of operating system, programmodules, application programs, and/or program data, that are accessibleto and/or presently being operated on by the processor, may be containedin the RAM. In embodiments, the program modules and/or applicationprograms can comprise a lookup table, an algorithm such as an algorithmto identify for determining a concentration of an analyte over anextended concentration range, and a comparison tool, which provides theinstructions for execution of processor.

The analyzer 305 may further include a barcode reader for readinginformation from a patient's bar-coded wristband, from a barcode on asensing device 310 or from any other item (e.g., a box of sensingdevices, box of control fluids, etc.) used in conjunction with theanalyzer 305. Other such encoding arrangements can be used. For example,the analyzer 305 may also include (either alternatively or in additionto the barcode reader) a radio-frequency (RF) identification device thatis capable of identifying an RF tag that is contained on or in eachindividual sensing device or each box of devices. According to anotherexemplary embodiment of the present invention, one or more of theencoding arrangements may be based upon a binary coding pin array of thetype disclosed in, for example, U.S. Pat. No. 4,954,087, which isincorporated herein by reference in its entirety. The various encodingarrangements may convey relevant information such as, for example, theidentity of a specific device type, date and location of manufacture,manufacturing lot number, expiration date, a unique number associatedwith a device, coefficients for use by the analyzer 305 associated withthe calculation of blood or other sample parameters, and the like.

Sensing Device or Cartridge

In one embodiment, as shown in FIG. 4, a sensing device or cartridge 400comprises a top portion 405 (e.g., a cover) and a bottom portion 410(e.g., a base) in which are mounted at least one microfabricated sensorchip 415 with electrical contacts and a pouch 420 containing a fluid,e.g., a wash fluid. The at least one sensor chip 415 may be positionedin recessed region 418 and configured to generate electric signals basedon a concentration of specific chemical species in a fluid sample, e.g.,a blood sample from a patient. In some embodiments, the composition ofthe fluid in the pouch 420 is selected from the group consisting ofwater, calibrant fluid, reagent fluid, control fluid, wash fluid andcombinations thereof. A gasket 425 is situated between the top portion405 and the bottom portion 410 to bond them together and to define andseal several cavities and conduits within the cartridge 400. The gasket425 may cover substantially the entire area between the top portion 405and the bottom portion 410 of the cartridge 400, as shown in FIG. 4, ormay be localized over and between only predetermined structuralfeatures, e.g., the at least one sensor chip 415, of the cartridge 400(not shown). The gasket 425 may include apertures 430 to enablephysical, fluidic and/or gaseous communication between structuralfeatures of the top portion 405 and the bottom portion 410. The gasket425 may or may not have an adhesive surface, and may have an adhesivesurface on both sides thereof, i.e., forming a double-sided adhesivelayer.

As shown in FIGS. 5A-5J, in some embodiments, the sensing device orcartridge 500 (e.g., cartridge 400 as described with respect to FIG. 4)has a housing that comprises a top portion 502 (e.g., a cover) and abottom portion 504 (e.g., a base) formed of rigid and flexible zones ofmaterial. As shown in FIGS. 5A-5J, the rigid zones (non-shaded portions)of the cover 502 and the base 504 respectively are preferably each asingle contiguous zone; however, the molding process can provide aplurality of non-contiguous substantially rigid zones. The flexiblezones (shaded portions) of the cover 502 and the base 504 respectivelyare preferably a set of several non-contiguous zones. For example, theflexible zone around a displaceable membrane may be separate anddistinct from the flexible zone at a closeable sealing member.Alternatively, the flexible zones may comprise a single contiguous zone.

The sensing device or cartridge 500 further comprises a sealable sampleentry port 506 and a closable sealing member 508 for closing the sampleentry port 502, a sample holding chamber 510 located downstream of thesample entry port 506, a capillary stop 512, a sensor region 514, and awaste chamber 516 located downstream of the sensor region 508.Preferably, the cross-sectional area of a portion of the sample holdingchamber 510 decreases distally with respect to the sample entry port506, as shown by ramp 518 in FIG. 5H. A pouch (e.g., the pouch 420described with respect to FIG. 4) may be disposed in a recessed region520 and in fluid communication with a conduit 522 leading to the sensorregion 514, optionally via conduit 524. The pouch may be of the designdescribed in U.S. Pat. No. 5,096,669 or, more preferably, in U.S. Pat.No. 8,216,529, both of which are incorporated herein by reference intheir entireties. Recessed region 520 preferably includes a spike 525configured to rupture the pouch, upon application of a force upon thepouch, for example, by reader or analyzer (e.g., analyzer 305 asdescribed with respect to FIG. 3). Once the pouch is ruptured, thesystem is configured to deliver the fluid contents from the pouch intoconduit 522. Movement of the fluid into the conduit 522 and to thesensor region 514 and/or within the conduit 524 may be effected by apump, e.g., a pneumatic pump connected to the conduit(s) 522 or 524.Preferably, the pneumatic pump comprises a displaceable membrane 526formed by a portion of a flexible zone 527 of the housing formed over arecessed region or airbladder 528. In the embodiment shown in FIGS.5A-5J, upon repeatedly depressing the displaceable membrane 526, thedevice pumps via conduits 524, 529, 530, and 531 causing fluid fromruptured pouch 206 to flow through the conduit 270, into the conduit 275and over the sensor region 230.

The closable sealing member 508, in some embodiments, includes a portionof the rigid zone that forms a sealing member 532, and a portion of theflexible zone that forms a seal 533. The sealing member 508 can rotateabout hinge 534 and engage the seal 533 with the sample entry port 506when in a closed position, thus providing an air-tight seal.Alternatively, an air-tight seal may be formed by contact of twoflexible materials, e.g., a thermoplastic elastomer (TPE) on TPE.Optionally, the sealable sample entry port 506 also includes a vent hole(not shown). In an alternative embodiment, a portion of the rigid zoneforms a sealing member, and a portion of the flexible zone forms aperimeter seal around the sample entry port, whereby the sealing membercan rotate about a hinge and engage the perimeter seal when in a closedposition, thus providing an air-tight seal. Alternatively, the perimeterseal may be formed by contact of two flexible materials. In yet anotherembodiment, the sealing member may include a slidable closure element asdescribed in pending U.S. Pat. No. 7,682,833, the entirety of which isincorporated herein by reference.

The sensor recess 514, in some embodiments, contains a sensor arraycomprising one or more sensors for one or more different analytes (orblood tests). For example, the sensor array may include an immunosensorand/or a magnetic immunosensor for one or more different analytes (orblood tests). The immunosensor may include a base sensor or sensingelectrode on a substantially planar chip (e.g., a microfabricated sensorchip such as the at least one sensor chip 415 described with respect toFIG. 4) where the sensing electrode is positioned in conduit 524 forreceiving a sample mixed with a reagent. The magnetic immunosensor mayinclude a base sensor or sensing electrode on a substantially planarchip (preferably the same sensor chip that includes the immunosensor)where the sensing electrode is positioned in the conduit 524 forreceiving a sample mixed with reagent that includes beads that can beattracted to a magnet, or respond to a magnetic field that is positionednear the magnetic immunosensor. In alternative embodiments, the sensorarray comprises a plurality of sensors for a plurality of differentanalytes (or blood tests). Accordingly, the cartridge 500 may have oneor more sensor recesses 514 each with at least one sensor.

The analytes/properties to which the sensors respond may be selectedfrom among pH, pCO₂, pO₂, glucose, lactate, creatinine, urea, sodium,potassium, chloride, calcium, magnesium, phosphate, hematocrit,prothrombin time (PT), activated partial thromboblastin time (APTT),activated clotting time (ACT), D-dimer, prostate-specific antigen (PSA),creatine kinase-MB (CKMB), brain natriuretic peptide (BNP), troponin I(TnI), cardiac traponin (cTnI), human chorionic gonadotrophin, troponinT, troponin C, myoglobin, and the like, and combinations thereof.Preferably, the analyte is tested in a liquid sample that is wholeblood, however other samples can be used including blood, serum, plasma,urine, cerebrospinal fluid, saliva and amended forms thereof. Amendmentscan include dilution, concentration, addition of regents such asanticoagulants and the like. Whatever the sample type, it can beaccommodated by the sample entry port 502 of the cartridge 500.

The cartridge 500 may further comprise a portion of the flexible zone536 positioned over the recessed region 520 that is configured for beingactuated upon like a pump to apply pressure within the recessed region520. In some embodiments, the flexible zone 536 may include a genericsymbol description to indicate to the user that pressure should not beapplied to the flexible zone 536 by the user. For example, the symbolmay comprise an embossed circle with a crossbar. The portion of theflexible zone 536 provides a surface that can accommodate an actuatorfeature of the analyzer (e.g., analyzer 305 as described with respect toFIG. 3) to apply a force and burst the underlying pouch in the recessedregion 520. The thickness of the plastic in the flexible zone 536 may bepreferably from about 200 to about 800 μm, for example about 400 μm.Essentially, the flexible zone 536 should be sufficiently thin to flexeasily, but sufficiently thick to maintain physical integrity and nottear.

Sensor and Chip Designs

In one embodiment, a microfabricated sensor chip (e.g., the at least onesensor chip 415 described with respect to FIG. 4) comprises at least onesensor or transducer (e.g., a working electrode or optical detector).For example, the microfabricated sensor chip may comprise a pair ofsensors comprising a first sensor (e.g., a low-end sensitivity sensor)and optionally a second sensor (e.g., a high-end sensitivity sensor). Insome embodiments, the sensors may be fabricated as adjacent structures,respectively, on a silicon chip.

In various embodiments, the sensors may be formed as electrodes withgold surfaces coated with a photo defined polyimide layer that includesopenings to define a grid of small gold electrodes (e.g., a goldmicroarray electrode) at which an electroactive species may be oxidized.For example, wafer-level micro-fabrication of a preferred embodiment ofthe sensor chip may be achieved as shown in FIG. 6A. A non-conductingsubstrate 600 having a planar top and bottom surface may be used as abase for the sensor chip. A conducting layer 602 may be deposited on thesubstrate 600 by conventional means, e.g., conductive printing, ormicro-fabrication technique known to those of skill in the art to format least one transistor. The conducting layer 602 may comprise a noblemetal such as gold, platinum, silver, palladium, iridium, or alloysthereof, although other unreactive metals such as titanium and tungstenor alloys thereof may also be used, as many non-metallic electrodes ofgraphite, conductive polymer, or other materials may also be used. Themicrofabricated sensor chip may also comprise an electrical connection603 that connects the electrode to a conductive pin such as a temporaryelectrical connector.

In some embodiments, the sensors may comprise an array of 5-10 μm noblemetal disks, e.g., 7 μm noble metal disks, on 15 μm centers. The arrayof noble metal disks or electrodes may cover a region, e.g., a circularregion, approximately 300 to 900 μm in diameter, optionally 400-800 μmor about 600 μm in diameter, and may be formed by photo-patterning athin layer of polyimide or photoresist of thickness up to 1.5 μm over asubstrate made from a series of layers comprising Si, SiO₂, TiW, and/orAu, or combinations thereof. In some embodiments, the electrodes have aworking area of about 130,000 to 300,000 sq μm (i.e., a microelectrode),the volume of sample directly over the electrodes may be about 0.1-0.3and the volume of the sample over the sensor chip may be 1-3 μL. Inaccordance with these aspects of the present invention, the conduit(e.g., the conduit 524 described with respect to FIG. 5A) in a region ofthe electrodes (e.g., the one or more sensor recesses 514 described withrespect to FIGS. 5A-5J) has a volume to sensor area ratio of less thanabout 64, to about 1 square mm, preferably less than about 50 mm toabout 2 square mm, more preferably less than about 100 μm to about 500square μm. Accordingly, the array of electrodes affords high collectionefficiency of a detectable moiety that is an electroactive species witha reduced contribution from any electrochemical background currentassociated with the capacitance of the exposed metal. In particular,openings in the insulating polyimide or photoresist layer define aregion of the noble metal electrodes at which the electroactive species,e.g., 4-aminophenol, may be oxidized such as in a two electron permolecule reaction.

Micro-fabrication techniques (e.g., photolithography and plasmadeposition) may be utilized for construction of the multilayered sensorstructures in confined spaces. For example, methods formicro-fabrication of electrochemical immunosensors on silicon substratesare disclosed in U.S. Pat. No. 5,200,051, which is hereby incorporatedby reference in its entirety, and include, for example, dispensingmethods, methods for attaching substrates and reagents to surfacesincluding photoformed layers, and methods for performing electrochemicalassays.

As shown in FIG. 6B, in some embodiments, a microfabricated extendedrange sensor chip 604 includes a first sensor 605 (e.g., a low-endsensitivity amperometric sensor) and optionally a second sensor 610(e.g., a high-end sensitivity amperometric sensor). The first and secondsensors 605, 610 may be fabricated as adjacent structures, respectively,on sensor chip 604. However, in order for the sensor chip 604 todetermine accurate analyte concentrations, the low-end sensitivitysensor 605 may be sufficiently spaced from the high-end sensitivitysensor 610. For example, at low to medium concentrations of analyte, thehigh-end sensitivity sensor 610 may generate a high amperometric signaldue to the high concentration of label reagent being attached toantibody coated magnetic beads. In embodiments in which the labelreagent uses an enzyme to cleave a substrate generating an electroactivespecies, the high concentration of the electroactive species at thehigh-end sensitivity sensor 610 can move along the sensor chip 604 andgenerate an amperometric signal on the low-end sensitivity sensor 605.Alternatively, the low concentration of the electroactive species at thelow-end sensitivity sensor could also move along the sensor chip andgenerate an amperometric signal on the high-end sensitivity sensor. Themagnitude of this crosstalk between the sensors depends on many factorsand can display variability between sensing device runs causingincreased imprecision on the amperometric reading of the low-endsensitivity sensor and/or the high-end sensitivity sensor. Accordingly,to reduce the crosstalk between sensors it may be beneficial in certainembodiments to space the two sensors from one another by a predetermineddistance.

The first sensor 605 and the second sensor 610 are spaced apart from oneanother at a predetermined distance “x”. For example, the first sensor605 may be spaced at least 0.03 mm, preferably at least 0.06 mm from thesecond sensor 610. The first sensor 605 may be connected via wiring 615to a first conductive pin 620 (e.g., temporary electrical connector) andthe second sensor 610 may be connected via wiring 625 to a secondconductive pin 630 (e.g., temporary electrical connector). In someembodiments, the first sensor 605 may be configured as an immunosensor(e.g., a low-end sensitivity amperometric sensor) and the second sensor610 may be configured as a magnetic immunosensor (e.g., a high-endsensitivity amperometric sensor) both of which are formed on the singlesensor chip 604 and positioned within one or more conduits of the pointof care test cartridge. Although it is shown in FIG. 6B that the secondsensor 610 is placed upstream from the first sensor 605, it should beunderstood that alternative embodiments of the present inventioncontemplate having the second sensor 610 placed downstream from thefirst sensor 605.

As illustrated in FIG. 6B, the first sensor 605 may be constructed withan array of metal disks or electrodes that cover a circular region in afirst area of the sensor chip 604 and the second sensor 610 may beconstructed with an array of metal disks or electrodes that cover acircular region in a second area of the sensor chip 604. The design andarrangement of the first and second sensors 605 and 610 on the sensorchip 604 are preferably selected based on printing and performancecharacteristics (e.g., minimize cross-talk between the sensors) for eachof the first and second sensors 605 and 610. However, it should beunderstood to those of ordinary skill in the art that any design orarrangement for the sensors is contemplated without departing from thespirit and scope of the present invention. Furthermore, although thefirst and second sensors 605 and 610 in the example in FIG. 6B aredescribed herein as amperometric sensors, other electrochemicalprocesses or optical processes which use other electrochemical oroptical sensors, e.g., optical wave guides and charge-coupled device(CCD) camera chips, can be used. For example, a potentiometric sensormay be used to detect ion species such as Na⁺ or K⁺.

As described herein, the first and second sensors 605 and 610 may beformed as electrodes with gold surfaces that are exposed (e.g., nopolyimide or photoresist covering) to the inside environment of theconduit and configured to directly contact a biological sample disposedwithin the conduit. The wirings 615 and 620 may be formed with goldsurfaces that are coated with a photo defined polyimide or photoresistlayer such that the wirings 615 and 620 are insulated from exposure tothe biological sample disposed within the conduit. The wirings 615 and620 may be formed comprising containment ring structures 635 and 640. Insome embodiments, the containment ring structure 635 for the firstsensor 605 may be configured to contain capture antibodies immobilizedon or near the surface of the electrodes. For example, the captureantibodies (as discussed herein) may be deposited onto at least aportion of the first sensor 605 within the containment ring structure635. The wirings 615 and 620 terminate at the first conductive pin 620and the second conductive pin 630 respectively, which are used to makecontact with a connector in an analyzer or cartridge reader (e.g., ani-STAT® cartridge reader as described in U.S. Pat. No. 4,954,087, theentirety of which is incorporated herein by reference).

In various embodiments, the first sensor 605 is an immunosensorpositioned in the conduit for receiving a biological sample mixed withan antibody-enzyme conjugate that is configured to bind to a targetanalyte within the biological sample. The first sensor 605 may beconfigured to detect an enzymatically produced electroactive species(e.g., 4-aminophenol) from the reaction of a substrate (e.g.,4-aminophenylphosphate) with the antibody-enzyme conjugate (e.g., one ormore antibodies bound to alkaline phosphatase (ALP)). In accordance withthese aspects, the first sensor 605 contains a capture region or regionscoated with capture antibodies 645 that are configured to bind to atarget analyte bound to an antibody-enzyme conjugate. The capture region645 may be defined by the containment ring structure 635. In someembodiments, the containment ring structure 635 is a hydrophobic ring ofpolyimide or another photolithographically produced layer. Amicrodroplet or several microdroplets (approximately 5-40 nL in size)containing capture antibodies in some form, for example bound to beadsor microspheres, may be dispensed on the surface of the first sensor605. The photodefined ring structure 635 contains this aqueous dropletallowing the capture region 645 to be localized to a precision of a fewmicrons. The capture region 645 can be made from 0.03 to roughly 2 mm²in size. The upper end of this size is limited by the size of theconduit and sensor chip 604 in present embodiments, and is not alimitation of the invention.

In some embodiments, a portion of the sensor chip 604 (e.g., a topsurface of the substrate), a wall of the conduit (e.g., the conduit 524described with respect to FIG. 5A), and/or a wall of the sample chamber(e.g., the sample chamber 510 described with respect to FIGS. 5G and 5H)can be coated with one or more dry reagents to amend the biologicalsample. For example, the sensor chip 604 may include a reagent region650 coated with an antibody-enzyme conjugate for an analyte of interest.The reagent region 650 may be defined by a containment ring structure655. In some embodiments, the containment ring structure 655 is ahydrophobic ring of polyimide or another photolithographically producedlayer. A microdroplet or several microdroplets (approximately 5-40 nL insize) or a series of about a 100 nanodroplets (approximately 50 to 1000pL in size) containing the antibody-enzyme conjugate in some form may bedispensed or printed on the surface of the sensor chip 604. Thephotodefined ring structure 655 contains this aqueous droplet allowingthe reagent region 650 to be localized to a precision of a few microns.The reagent region 650 can be made from 0.03 to roughly 2 mm² in size.The upper end of this size is limited by the size of the conduit andsensor chip 604 in present embodiments, and is not a limitation of theinvention.

The biological sample or a fluid may be passed at least once over thedry reagent, e.g., the reagent region 650 to dissolve the reagent withinthe biological sample or fluid. Reagents used to amend biologicalsamples or fluid within the cartridge may include the antibody-enzymeconjugate, magnetic beads coated with capture antibodies, or blockingagents that prevent either specific or non-specific binding reactionsamong assay compounds. Within a segment of the biological sample orfluid, the reagent can be preferentially dissolved and concentratedwithin a predetermined region of the segment. This is achieved throughcontrol of the position and movement of the segment. Thus, for example,if only a portion of a segment, such as the leading edge, isreciprocated over the reagent, then a high local concentration of thereagent can be achieved close to the leading edge. Alternatively, if ahomogenous distribution of the reagent is desired, for example if aknown concentration of a reagent is required for a quantitativeanalysis, then further reciprocation of the sample or fluid will resultin mixing and an even distribution.

In the preferred embodiments of the present invention, the analyzerapplies a potential via the first conductive pin 620 to the first sensor605 and a reference electrode, and measures current changes generated byoxidation current from the substrate as an electrochemical signal. Theelectrochemical signal being proportional to the concentration of theanalyte in the biological sample. The first sensor 605 has an appliedpotential of approximately +0 mV to 90 mV, e.g., +60 mV versus thereference electrode and, in another preferred embodiment, the firstsensor 605 has an applied potential of approximately +40 mV versus thereference electrode. The signal generated by the enzyme reaction productat approximately +10 mV is distinguishable from the signal generated bythe unreacted substrate at approximately +200 mV. It should be notedthat the exact voltages used to amperometrically detect the substrateand the analyte will vary depending on the chemical structure of thesubstrate. It is important that the difference in the voltages used todetect the substrate be great enough to prevent interference between thereadings.

In various embodiments, the second sensor 610 is a magnetic immunosensorpositioned in the conduit for receiving a biological sample mixed withbeads that can be attracted to a magnet, or respond to a magnetic field.The beads are coated with capture antibodies that are configured to bindto the target analyte bound to the antibody-enzyme conjugate, e.g., theantibody-enzyme conjugate disposed in reagent region 650 andsubsequently dissolved in the biological sample. The second sensor 610may be configured to detect an enzymatically produced electroactivespecies (e.g., 4-aminophenol) from the reaction of a substrate (e.g.,4-aminophenylphosphate) with the antibody-enzyme conjugate (e.g., one ormore antibodies bound to alkaline phosphatase (ALP)). In accordance withthese aspects, a high-field magnet, e.g., a permanent magnet or anelectromagnet, may be positioned proximate to the sensor chip 604 (e.g.,below) or incorporated into the sensor chip 604, to generate a magneticfield for attracting the beads mixed with the biological sample in theconduit to a location substantially proximate to the second sensor 610.The magnetic field is localized around the second sensor 610 based onthe predetermined distance “x” between the first sensor 605 and thesecond sensor 610, and functions to substantially retain the beads at ornear the surface of the second sensor 610 during removal of unboundsample and washing of the electrodes.

The high-field magnet of the present invention may include any materialthat provides a high magnetic field (e.g., greater than about 0.1 Tesla,greater than 0.4 Tesla or greater than 1 Tesla). The magnetic field canbe measured, for example, as a remnant field on a substantially flatsurface area of the magnet. In some embodiments, the high-field magnetis comprised of a material such as neodymium iron boron alloy (NdFeB)alloy (e.g., Nd₂Fe₁₄B), or ferrite or aluminum nickel cobalt (AlNiCo),which typically exhibit fields of greater than 0.1 Tesla, e.g., greaterthan 0.5 Tesla or from 0.1 to 1 Tesla. In other embodiments, thehigh-field magnet is comprised of alloys of rare earth elements (e.g.,neodymium alloys and samarium cobalt (SmCo) alloys), which exhibitfields of greater than 0.1 Tesla, e.g., greater than 1.2 Tesla orgreater than 1.4 Tesla. In alternative embodiments, the high-fieldmagnet comprises an electromagnet in which the magnetic field isproduced by the flow of electric current. The electric current may beprovided by an analyzer, in which the sensing device is inserted andwith which the sensing device is in electrical contact.

The high-field magnet can be provided proximate to the sensor chip 604(e.g., below) or incorporated into the sensor chip 604 using a number oftechniques as described in detail herein. In some embodiments, thesecond sensor 610 comprises a sensing electrode on a substantiallyplanar substrate and a bulk permanent high-field magnet positionedproximate to the electrode (e.g., below or on the opposite side of thesensor chip 604). In certain preferred embodiments, the bulk permanenthigh-field magnet is positioned in the housing (e.g., cut out or trenchin the rigid zone of the cartridge) of the sensing device. For example,the bulk permanent high-field magnet may be positioned within the baseof the cartridge housing (e.g., non-coplanar with the sensor chip). Inother embodiments, the high-field magnet is positioned adjacent to orwithin the analyzer, in which the sensing device is inserted. The bulkhigh-field permanent magnet may be substantially cylindrical, having adiameter in the range of about 0.1 mm to about 5 mm and a length ofabout 0.1 mm to about 5 mm, and is positioned to yield an “eventhorizon” (as defined herein) in the conduit suitable for bead capturewithin a short period of time (e.g., 1-5 minutes). The conduit generallyhas a height of about 0.2 mm to about 5 mm and a width of about 0.2 mmto about 5 mm, and either a uniform or non-uniform cross-sectional area.Alternatively, the bulk magnet shape may be in the form of a square,rectangle, oval, flake, pyramid, sphere, sub-sphere, or other shapedform.

In alternative embodiments, the second sensor 610 comprises a sensingelectrode on a substantially planar substrate and a magnetized layer(e.g., microfabricated magnetic layer). The magnetized layer may beincluded on (e.g., positioned over, directly attached, coated orpatterned onto any surface of the sensor chip 604) or embedded into thechip (e.g., positioned within the chip, integral to the chip). Thisconfiguration attracts the magnetically susceptible beads substantiallyproximate to or on the sensing electrode and substantially retains themat the sensing electrode during removal of unbound sample and washing ofthe sensing electrode.

The magnetized layer may be a composite material formed from aparticulate magnetic material capable of sustaining a high-fieldpermanent magnetic field, e.g., a NdFeB alloy, in a binder or supportmatrix (e.g., a thermal setting ink, a polyimide, polyvinyl alcohol(PVA) or thermoplastic equivalent). In addition to thermal setting ink,polyimide, PVA and thermoplastic equivalents, two-part chemically curedepoxy resins, kapton and the like may be used as the binder for fixingthe particulate magnetic material to the sensor chip. In someembodiments, the binder is comprised of a thermal setting ink such as asolvent based encapsulant screen printing ink or an acrylic acid polymerin a solvent. In alternative embodiments, the binder is comprised ofother photoformed matrix materials. The methods of curing the compositematerial may be based on a photo-initiated, thermally initiated or achemically initiated process. The composite material is not limited byviscosity and can include any viscosity suitable for application. Insome embodiments, the composite material has a viscosity ranging from0.3 to 300,000 CPS, e.g., from 100 to 100,000 CPS or from 1,000 to10,000 CPS. The magnetic particles in the composite material of certainembodiments have an average particle size from 0.01 μm to 100 μm, e.g.,from 0.1 μm to 10 μm or from 3 μm to 7 μm.

The composite material can be applied in a variety of locations in or onthe sensing device (e.g., to the front side or backside of a wafer orchip, electrode, housing, reader, etc.). For example, in someembodiments, the composite material is applied to the sensor chip in apatterned manner (e.g., using a mask). In other embodiments, thecomposite material is applied to the sensing electrode. In otherembodiments, the composite material is applied in a magnetized layerbelow the sensing electrode. Prior to the application of the magnetizedlayer, the magnetized layer may or may not be magnetized. However, afterthe application, the magnetic layer preferably is magnetized to providedirectionality to the magnetic field generated by the magnetized layer.

In some embodiments, a portion of the sensor chip 604 (e.g., a topsurface of the substrate), a wall of the conduit (e.g., the conduit 524described with respect to FIG. 5A), and/or a wall of the sample chamber(e.g., the sample chamber 510 described with respect to FIGS. 5G and 5H)can be coated with one or more dry reagents to amend the biologicalsample. For example, the sensor chip 604 may include a reagent region660 coated with magnetic beads having capture antibodies for an analyteof interest. The reagent region 660 may be defined by a containment ringstructure 665. In some embodiments, the containment ring structure 665is a hydrophobic ring of polyimide or another photolithographicallyproduced layer. A microdroplet or several microdroplets (approximately5-40 nL in size) containing the antibody-enzyme conjugate in some formmay be dispensed or printed on the surface of the sensor chip 604. Thephotodefined ring structure 665 contains this aqueous droplet allowingthe reagent region 660 to be localized to a precision of a few microns.The reagent region 665 can be made from 0.03 to roughly 2 mm² in size.The upper end of this size is limited by the size of the conduit andsensor chip 604 in present embodiments, and is not a limitation of theinvention. Although it is shown in FIG. 6B that the reagent region 660is placed upstream from the reagent region 650, it should be understoodthat alternative embodiments of the present invention contemplate havingthe reagent region 660 placed downstream from the reagent region 650.

The biological sample or a fluid may be passed at least once over thedry reagent, e.g., the reagent region 660 to dissolve the reagent withinthe biological sample or fluid. Reagents used to amend biologicalsamples or fluid within the cartridge may include the antibody-enzymeconjugate, magnetic beads coated with capture antibodies, or blockingagents that prevent either specific or non-specific binding reactionsamong assay compounds. Within a segment of the biological sample orfluid, the reagent can be preferentially dissolved and concentratedwithin a predetermined region of the segment. This is achieved throughcontrol of the position and movement of the segment. Thus, for example,if only a portion of a segment, such as the leading edge, isreciprocated over the reagent, then a high local concentration of thereagent can be achieved close to the leading edge. Alternatively, if ahomogenous distribution of the reagent is desired, for example if aknown concentration of a reagent is required for a quantitativeanalysis, then further reciprocation of the sample or fluid will resultin mixing and an even distribution.

In the preferred embodiments of the present invention, the analyzerapplies a potential via the second conductive pin 630 to the secondsensor 610 and a reference electrode, and measures current changesgenerated by oxidation current from the substrate as an electrochemicalsignal. The electrochemical signal being proportional to theconcentration of the analyte in the biological sample. The second sensor610 has an applied potential of approximately +0 mV to 90 mV, e.g., 60mV versus the reference electrode and, in another preferred embodiment,the first sensor 605 has an applied potential of approximately +40 mVversus the reference electrode. The signal generated by the enzymereaction product at approximately +10 mV is distinguishable from thesignal generated by the unreacted substrate at approximately +200 mV. Itshould be noted that the exact voltages used to amperometrically detectthe substrate and the analyte will vary depending on the chemicalstructure of the substrate. It is important that the difference in thevoltages used to detect the substrate be great enough to preventinterference between the readings.

In some embodiments, the sensor chip 604 may further include aconductometric sensor 670 (e.g., hematocrit sensors). The conductometricsensor 670 is configured to determine biological sample arrival and/ordeparture at the reagent regions 650 and 660 and biological samplearrival and/or departure at the first and second sensors 605 and 610.More specifically, the conductometric sensor 670 lie perpendicular to alength of the conduit or sensor conduit, and an electrical resistancebetween pairs of electrodes for the sensor may be used to monitor arelative position of a fluid front of the biological sample. At theextremes, an open circuit reading indicates that the biological samplehas been pushed off the reagent regions 650 and 660 and a closed circuitreading indicates the reagent regions 650 and 660 are covered with thebiological sample.

As shown in FIG. 6B, the conductometric sensor 670 may comprise at leasttwo electrodes 675 and 680 (i.e., electrode pair) positioned downstreamof the first and second sensors 605 and 610. The electrodes 675 and 680may be connected via wirings 685 and 690 to a conductometric low pin 692and an AC source or conductometric high pin 695, respectively (e.g.,temporary electrical connectors). The wirings 685 and 690 may be formedwith a gold surface that is coated with a photo defined polyimide orphotoresist layer such that the wirings 685 and 690 are insulated fromexposure to the biological sample disposed within the conduits. As such,in some embodiments, the biological sample or fluid reaches theelectrode pair in a conduit (e.g., prior to arriving at the first andsecond sensors 605 and 610), then subsequently arrives at the first andsecond sensors 605 and 610 (e.g., after departing the reagent regions650 and 660).

As shown in FIG. 7, in alternative embodiments, a microfabricated sensorchip 700 includes a first sensor 705 (e.g., a low-end sensitivityamperometric sensor) and optionally a second sensor 710 (e.g., ahigh-end sensitivity amperometric sensor), as similarly described withrespect to FIG. 6B. However, as illustrated in FIG. 7, the first sensor705 may be constructed with an array of metal disks or electrodes thatcover a circular region in a first area of the sensor chip 700 and thesecond sensor 710 may be constructed with an array of metal disks orelectrodes that cover a square or elongated region 715 in a second areaof the sensor chip 700. The square or elongated region 715 in a secondarea of the sensor chip 700 provides a larger surface area for themagnet or magnetic field to capture the beads coated with captureantibodies dispersed with the biological sample, as the biologicalsample passes through the conduit over the sensors. As should beunderstood, the microfabricated sensor chip 700 may include one or moreof the same additional features such as the reagent regions and theconductometric sensor as described with respect to the sensor chip 604and FIG. 6B.

As shown in FIGS. 8A and 8B, in other embodiments designed to lower thecrosstalk between the two sensors, a microfabricated extended rangesensor chip 800 (e.g., the at least one sensor chip 415 described withrespect to FIG. 4) is provided for that comprises a pair of sensorscomprising a first sensor (e.g., a low-end sensitivity sensor) and asecond sensor (e.g., a high-end sensitivity sensor) with a scavengingelectrode provided between the sensors. In some embodiments, the firstand second sensors may be fabricated as adjacent structures,respectively, on a silicon chip. However, in order for the extendedrange sensor chip to determine accurate analyte concentrations, thelow-end sensitivity sensor may be sufficiently isolated from thehigh-end sensitivity sensor. For example, at low to mediumconcentrations of analyte, the high-end sensitivity sensor may generatea high amperometric signal due to the high concentration of the labelreagent being attached to the antibody coated magnetic beads. Inembodiments in which the label reagent uses an enzyme to cleave asubstrate generating an electroactive species, the high concentration ofthe electroactive species at the high-end sensitivity sensor can movealong the sensor chip and generate an amperometric signal on the low-endsensitivity sensor. Alternatively, the low concentration of theelectroactive species at the low-end sensitivity sensor could also movealong the sensor chip and generate an amperometric signal on thehigh-end sensitivity sensor. The magnitude of this crosstalk between thesensors depends on many factors and can display variability betweensensing device runs causing increased imprecision on the amperometricreading of the low-end sensitivity sensor and/or the high-endsensitivity sensor. Accordingly, it may be beneficial in certaincircumstances to lower the crosstalk between the two sensors in theextended range sensor chip using a scavenging electrode.

The microfabricated sensor chip 800 includes a first sensor 805 (e.g., alow-end sensitivity amperometric sensor) and a second sensor 810 (e.g.,a high-end sensitivity amperometric sensor), as similarly described withrespect to FIG. 6B. However, the first sensor 805 and the second sensor810 are spaced apart from one another at an increased distance “y” ascompared to the sensor chip 604 shown in FIG. 6B. For example, the firstsensor 805 may be spaced at least 0.2 mm, preferably at least 0.5 mmfrom the second sensor 810. The first sensor 805 may be connected viawiring 815 to a first conductive pin 820 (e.g., temporary electricalconnector) and the second sensor 810 may be connected via wiring 825 toa second conductive pin 830 (e.g., temporary electrical connector). Insome embodiments, the first sensor 805 may be configured as animmunosensor (e.g., a low-end sensitivity amperometric sensor) and thesecond sensor 810 may be configured as a magnetic immunosensor (e.g., ahigh-end sensitivity amperometric sensor) both of which are formed onthe single sensor chip 800 and positioned within one or more conduits ofthe point of care test cartridge.

As shown in FIGS. 8A and 8B, the increased spacing “y” between the firstsensor 805 and the second sensor 810 allows for a scavenging electrode835 to be positioned between the first sensor 805 and the second sensor810. Specifically, the design and arrangement of the first and secondsensors 805 and 810 on the sensor chip 800 are selected to allow for theaddition of the scavenging electrode 835 between the first sensor 805and the second sensor 810. The scavenging electrode 835 is configured tooxidize the electroactive species generated at the second sensor 810 sothat high signals at the second sensor 810 do not result in significantcrosstalk at the first sensor 805 and/or low signals at the first sensor805 do not result in significant crosstalk at the second sensor 810. Forexample, the scavenging electrode is configured to (i) preventelectroactive species generated in a region of the second sensor fromdiffusing to the first sensor, (ii) prevent electroactive speciesgenerated in a region of the second sensor from being transported to thefirst sensor, (iii) prevent electroactive species generated in a regionof the second sensor from being detected at the first sensor, (iv)prevent electroactive species generated in a region of the first sensorfrom diffusing to the second sensor, (v) prevent electroactive speciesgenerated in a region of the first sensor from being transported to thesecond sensor, and/or (vi) prevent electroactive species generated in aregion of the first sensor from being detected at the second sensor.

In some embodiments, as shown in FIG. 8A, the scavenging electrode 835is connected via wiring 840 to the second sensor 810. In alternativeembodiments, as shown in FIG. 8B, the scavenging electrode 1735 isconnected via wiring 845 to a conductometric low pin 850 (e.g.,temporary electrical connector for the conductivity sensor). Bothconfigurations of the scavenging electrode 835 are designed to minimizecrosstalk while resulting in low impact on the signal generated at thesecond sensor 810 and the signal generated at the first sensor 805. Asshould be understood, the microfabricated sensor chip 800 may includeone or more of the same additional features such as the reagent regionsand the conductometric sensor as described with respect to the sensorchip 604 and FIG. 6B.

Magnetic Immunosensor Configurations

FIGS. 9A-9C show three exemplary embodiments of a magnetic immunosensor(e.g., a high-end sensitivity amperometric sensor 610 as described withrespect to FIG. 6B) where the magnetic component is directly integratedinto the base or cartridge housing of the sensing device. In eachembodiment shown in FIGS. 9A-9C, the sensor chip 900 includes animmunosensor 905 disposed in a conduit 910 and positioned on a surfaceof a substrate 915 above a high-field magnet 920. The high-field magnet920 may be cylindrical with a length of from 1 mm to 10 mm, e.g., from 2mm to 5 mm, preferably about 3 mm, and a diameter of from 0.1 mm to 5mm, e.g., from 0.5 mm to 2 mm. In FIGS. 9A-9C, the high-field magnet 920has diameters of about 1 mm, about 0.5 mm and about 0.3 mm,respectively. The high-field magnet 920 is within the base or cartridgehousing 925 (e.g., a bottom portion or base 504 as described withrespect to FIGS. 5A-5J) of the sensing device and optionally is abuttedto the underside of the sensor chip 900, which preferably has athickness of from about 0.2 mm to 5 mm, e.g., from 0.5 mm to 2 mm orpreferably about 1 mm. In accordance with these aspects, a high-fieldmagnet, e.g., a permanent magnet or an electromagnet, may be positionedproximate to the sensor chip 900 (e.g., below), for attractingmagnetically susceptible beads in the conduit substantially proximate toor on the sensor 905.

As shown in FIG. 10, a device 1000 for detecting an analyte in abiological sample in accordance with the some aspects of the presentinvention comprises a substrate 1005 including a planar top and bottomsurface 1010, 1015, a first electrochemical sensor 1020 (e.g., a low-endsensitivity amperometric sensor) positioned on the top surface 1010 ofthe substrate 1005, and a second electrochemical sensor 1025 (e.g., ahigh-end sensitivity amperometric sensor) positioned on the top surface1010 of the substrate 1005 and adjacent to the first electrochemicalsensor 1020. In some embodiments, the substrate 1005 is disposed withina conduit 1027 of the device 1000. The substrate 1005 may be comprisedof a base material selected from the group consisting of silicon, glass,and plastic. The first electrochemical sensor 1020 may include animmobilized layer of antibody 1030 configured to bind to an analyte suchas cTnI. The first electrochemical sensor 1020 and the secondelectrochemical sensor 1025 may comprise a gold microarray electrode andhave a diameter from about 100 μm to about 500 μm or from about 200 μmto about 1500 μm.

The device 1000 further includes (i) a first reagent region 1035 on thesubstrate 1005 coated with an antibody-enzyme conjugate for the analyte,and/or (ii) a second reagent region 1040 on the substrate 1005 coatedwith magnetic beads having capture antibodies for the analyte. Thereagent regions 1035, 1040 may be defined by a containment ringstructure 1045, 1050, respectively. In other embodiments, the reagentregions 1035, 1040 may be located on the conduit 1027 (e.g., the conduit524 described with respect to FIG. 5A), and/or in the sample chamber(e.g., the sample chamber 510 described with respect to FIGS. 5G and5H).

The device 1000 further includes a housing 1055 that supports thesubstrate 1005. The housing 1055 having an opening or trench 1060 thatextends to a region 1065 in the housing below the second electrochemicalsensor 1025. The opening or trench 1060 comprises a high-field magnet1070 (e.g., bulk permanent high-field magnet) that optionally abuts theplanar bottom surface 1015 of the substrate 1005. The high-field magnet1070 has a shape (e.g., a shape that is substantially triangular,trapezoid, column, rectangle, square, circular, pyramid, etc.(substantially in this context would be understood by those of ordinaryskill in the art to mean that visually the shape is by and largetriangular, trapezoid, column, rectangle, square, circular, pyramid,etc)) that fits within the opening or trench 1060. Moreover, thehigh-field magnet 1070 generates a magnetic field 1075 that is aligned(e.g., on a same vertical plane) with respect to the secondelectrochemical sensor 1025 and/or orthogonal to a horizontal plane ofthe top surface 1010 of the substrate 1005. The magnetic field 1075 isconfigured to focus and attract the magnetic beads onto a surface of thesecond electrochemical sensor 1025 once the magnetic beads are mixedwith the biological sample.

In alternative embodiments, a magnetic immunosensor (e.g., a high-endsensitivity amperometric sensor 610 as described with respect to FIG.6B) is provided where the magnetic component is directly integrated intothe sensor manufacture, rather than being a separate component (e.g.,bulk permanent high-field magnet) requiring assembly into the base orcartridge housing of the sensing device. The magnetized layer may beformed from a composite material, e.g., slurry, comprising a particulatemagnetic material capable of sustaining a high-field permanent magneticfield, e.g., a NdFeB alloy, in a binder or support matrix (e.g., apolyimide, polyvinyl alcohol (PVA) or thermoplastic equivalent). In oneembodiment, a mixture of photoformable polyvinyl alcohol (PVA) mixedwith ground Nd₂Fe₁₄B powder is printed onto a wafer using amicrodispensing apparatus of the type described in U.S. Pat. No.5,554,339, which is incorporated herein by reference in its entirety.The printed area may be a diameter of about 400 or from 200 to 600 μm.FIGS. 11A and 11B show exemplary immunosensors 1100 partially coveredwith a printed polyimide and NdFeB particle matrix 1105 leaving aportion 1110 of the perimeter of the magnetic immunosensors exposed.

In another embodiment, the slurry of magnetizable particles (e.g.,ground Nd₂Fe₁₄B powder) is deposited in a trench within thenon-conducting substrate or wafer of the microfabricated sensor chip.FIG. 12 illustrates trench forming process that comprises initiallyetching a non-conducting substrate 1200 (e.g., a silicon wafer) having asurface coating of photoresist 1205 with hydrofluoric acid (HF) and thenetching the substrate with hot potassium hydroxide (KOH) or trimethylammonium hydroxide (TMAH) to leave a trench 1210 of controlled profile(e.g., a shape that is substantially triangular, trapezoid, column,rectangle, square, circular, pyramid, etc. (substantially in thiscontext would be understood by those of ordinary skill in the art tomean that visually the shape is by and large triangular, trapezoid,column, rectangle, square, circular, pyramid, etc)) and dimensions(e.g., a depth and width of from about 5 μm to about 600 μm). A slurryof magnetizable particles (e.g., NdFeB alloy powder) in a thermoplasticmatrix (e.g., polyimide) is then microdispensed 1215 or spin-coated 1220into the trench 1210 to form a magnetized layer 1225 having asubstantially flat surface co-planar with the substrate 1200. Thesubstrate 1200 may be further processed, as described in jointly-ownedU.S. Pat. Nos. 7,419,821 and 7,723,099, which are incorporate herein byreference in their entireties, to provide an immunosensor array 1230over each etched trench 1210 on the substrate 1200. The immunosensorarray 1230 may be deposited directly on the magnetized layer 1225 asshown in (a) or over the magnetized layer 1225 as shown in (b).

As shown in FIG. 13, a device 1300 for detecting an analyte in abiological sample in accordance with the some aspects of the presentinvention comprises a substrate 1305 including a planar top and bottomsurface 1310, 1315, a first electrochemical sensor 1320 (e.g., a low-endsensitivity amperometric sensor) positioned on the top surface 1310 ofthe substrate 1305, and a second electrochemical sensor 1325 (e.g., ahigh-end sensitivity amperometric sensor) positioned on the top surface1310 of the substrate 1305 and adjacent to the first electrochemicalsensor 1320. In some embodiments, the substrate 1305 is disposed withina conduit 1327 of the device 1000. The substrate 1305 may be comprisedof a base material selected from the group consisting of silicon, glass,and plastic. The first electrochemical sensor 1320 may include animmobilized layer of antibody 1330 configured to bind to an antibodysuch as cTnI. The first electrochemical sensor 1320 and the secondelectrochemical sensor 1325 may comprise a gold microarray electrode andhave a diameter from about 100 μm to about 500 μm or from about 200 μmto about 1500 μm.

The device 1300 further includes (i) a first reagent region 1335 on thesubstrate 1305 coated with an antibody-enzyme conjugate for the analyte,and/or (ii) a second reagent region 1340 on the substrate 1005 coatedwith magnetic beads having capture antibodies for the analyte. Thereagent regions 1335, 1340 may be defined by a containment ringstructure 1345, 1350, respectively. In other embodiments, the reagentregions 1335, 1340 may be located on the conduit 1327 (e.g., the conduit524 described with respect to FIG. 5A), and/or in the sample chamber(e.g., the sample chamber 510 described with respect to FIGS. 5G and5H).

The device 1300 further includes an opening or trench 1355 in the bottomsurface 1315 of the substrate extending to a region 1360 in thesubstrate 1305 below the second electrochemical sensor 1325. The openingor trench 1355 comprises a composite material 1365 including a binder(e.g., the binder is comprised of polyimide or polyvinyl alcohol) and aparticulate magnetic material (e.g., the particulate magnetic materialis comprised of neodymium iron boron (NdFeB) alloy or aluminum nickelcobalt (AlNiCo) alloy) that optionally fills the opening or trench 1355.The composite material 1365 is configured to take on the shape of theopening or trench 1355 (e.g., a shape that is substantially triangular,trapezoid, column, rectangle, square, circular, pyramid, etc.(substantially in this context would be understood by those of ordinaryskill in the art to mean that visually the shape is by and largetriangular, trapezoid, column, rectangle, square, circular, pyramid,etc)).

In some embodiments, a shape of the opening or trench 1355 includes asubstantially triangular cross-section, a base 1370 of the substantiallytriangular cross-section is co-planar with the bottom surface 1315 ofthe substrate 1305, and an apex 1375 of the substantially triangularcross-section is below the second electrochemical sensor 1325. Theopening or trench 1355 may have a diameter from about 200 μm to about1500 μm, for example from 500 μm to 1000 μm. The substantiallytriangular cross-section shape of the opening may be selected from thegroup consisting of: a cone, a pyramid, a tetrahedron, a polygon ofconical form, and a V-shaped trench. The substantially triangularcross-section may extend through at least 75%, 90%, or 95% of a distancefrom the bottom surface 1315 to the top surface 1310 of the substrate1305. The composite material 1365 generates a magnetic field 1380 thatis aligned (e.g., on a same vertical plane) with respect to the secondelectrochemical sensor 1325 and/or orthogonal to a horizontal plane ofthe top surface 1310 of the substrate 1305. The magnetic field 1380 isconfigured to focus and attract the magnetic beads onto a surface of thesecond electrochemical sensor 1325 once the magnetic beads are mixedwith the biological sample.

Combined Immunoassay Methods

FIGS. 14-17 show exemplary flowcharts for performing the process stepsof the present invention. The steps of FIGS. 14-17 may be implementedusing the computing devices and systems described above with respect toFIGS. 1-13. Specifically, the flowcharts in FIGS. 14-17 illustrate thearchitecture, functionality, and operation of possible implementationsof the systems, methods and computer program products according toseveral embodiments of the present invention. In this regard, each blockin the flowcharts may represent a module, segment, or portion of code,which comprises one or more executable instructions stored onnon-transitory machine readable storage medium that when executed by oneor more processors (e.g., a processor of the analyzer) cause the one ormore processors to perform the specified logical function(s) within theone or more executable instructions. It should also be noted that, insome alternative implementations, the functions noted in the blocks mayoccur out of the order noted in the figure. For example, two blocksshown in succession may, in fact, be executed substantiallyconcurrently, or the blocks may sometimes be executed in the reverseorder, depending upon the functionality involved. It will also be notedthat each block of the flowchart illustrations, and combinations ofblocks in the flowchart illustrations, can be implemented by specialpurpose hardware-based systems that perform the specified functions oracts, or combinations of special purpose hardware and computerinstructions.

FIG. 14 illustrates a method 1400 (with reference to the sensing device500 as illustrated in FIGS. 5A-5J) of using a sensing device inaccordance with one embodiment of the invention. At step 1405, anunmetered biological sample may be introduced into a sample chamber(e.g., the sample chamber 510 described with respect to FIGS. 5G and 5H)of a sensing device, through a sample entry port (e.g., sealable sampleentry port 506 described with respect to FIGS. 5B and 5C). At step 1410,a capillary stop (e.g., capillary stop 512 described with respect toFIGS. 5G and 5H) may prevent passage of the sample into a first conduit(e.g., conduit 531 described with respect to FIG. 5A) at this stage, andthe sample chamber is filled with the sample. The capillary stop at theend of the sample chamber delimits a metered portion of the biologicalsample. At step 1415, a lid (e.g., closable sealing member 508 describedwith respect to FIGS. 5A and 5B) maybe closed to prevent leakage of thebiological sample from out of the sensing device. While the biologicalsample is within sample chamber, the biological sample may be optionallyamended at step 1420 with a compound or compounds (e.g., reagents suchas antibody-coated magnetically susceptible beads and enzyme-labeledantibody conjugate) present initially as a dry coating on the innersurface of the chamber.

At step 1425, the sensing device may be inserted into an analyzer (e.g.,analyzer 305 described with respect to FIG. 3) in accordance with someaspects of the present invention. At step 1430, insertion of the sensingdevice into the analyzer may activate a first pump (e.g., the portion ofthe flexible zone 536 as described with respect to FIGS. 5A and 5B) ormechanism that punctures a fluid-containing package when the package ispressed against a spike (e.g., spike 525 as described with respect toFIGS. 5G and 5H). Fluid (e.g., a substrate) may thereby expel into asecond conduit (e.g., conduit 522 as described with respect to FIGS. 5Gand 5H) that is in fluidic communication with the first conduit. Aconstriction in the second conduit prevents further movement of thefluid. At step 1435, operation of a second pump (e.g., displaceablemembrane 526 as described with respect to FIGS. 5A, 5B, 5G, and 5H) bythe analyzer applies pressure to an air-bladder of the sensing device,forcing air through a third conduit (e.g., conduit 529 as described withrespect to FIGS. 5G and 5H) and into the sample chamber at apredetermined location.

At step 1440, the metered portion of the biological sample is expelledthrough the capillary stop by air pressure produced within theair-bladder at step 1435 into the first conduit. At step 1445, thebiological sample is move forward within the first conduit to a portionof the first conduit (e.g., conduit 524 as described with respect toFIG. 5A) that is exposed to a sensor chip (e.g., sensor chip 604 asdescribed with respect to FIG. 6B) by air pressure produced within theair-bladder. Optionally at step 1450, the biological sample is amendedwith a compound or compounds (e.g., reagents such as antibody-coatedmagnetically susceptible beads and enzyme-labeled antibody conjugate)present initially as a dry coating on a portion of the sensor chip(i.e., one or more reagent regions). To facilitate the dissolution ofthe compound or compounds in the biological sample and/or promoteefficient sandwich formation on the magnetically susceptible beads, thebiological sample may be oscillated over the one or more reagent regionsby air pressure produced within the air-bladder. In one embodiment, anoscillation frequency of between about 0.2 Hz and about 5 Hz is used,most preferably about 0.7 Hz. At step 1455, the amended biologicalsample is move forward within the first conduit to a position over afirst sensor (e.g., a low-end sensitivity amperometric sensor) andoptionally a second sensor (e.g., a high-end sensitivity amperometricsensor) by air pressure produced within the air-bladder. Optionally atstep 1460, to facilitate trapping the magnetically susceptible beadswithin a magnetic field on or near a surface of the second sensor and/orpromote efficient sandwich formation on or near the surface of the firstsensor comprising a biolayer, the biological sample may be oscillatedover the first and second sensors by air pressure produced within theair-bladder. In one embodiment, an oscillation frequency of betweenabout 0.2 Hz and about 5 Hz is used, most preferably about 0.7 Hz.

At step 1465, the biological sample is displaced from the first conduitby further pressure applied to air-bladder, and the biological samplepasses to a waste chamber (e.g., waste chamber 516 as described withrespect to FIGS. 5A and 5G.). At optional step 1470, one or more airsegments (meniscus) may be produced within the first conduit by anysuitable means, including a passive means, an embodiment of which isdescribed in detail in U.S. Pat. No. 7,682,833, which is incorporatedherein by reference in its entirety, or an active means including atransient lowering of the pressure within the first conduit using thesecond pump whereby air is drawn into the first conduit through a flapor valve. The one or more air segments are extremely effective atclearing or rinsing the biological sample-contaminated fluid from thefirst conduit. For example, a leading and/or trailing edge of the one ormore air segments may be passed a number of times over the first andsecond sensors to rinse and resuspend extraneous material that may havebeen deposited from the biological sample. Extraneous material includesany material other than specifically bound analyte oranalyte/antibody-enzyme conjugate complex. However, in accordance withvarious embodiments, the clearing or rinsing step 1470 using the one ormore air segments is not sufficiently protracted or vigorous so as topromote substantial resuspension of the magnetically susceptible beadsor dissociation of specifically bound analyte or analyte/antibody-enzymeconjugate complex from the beads or the biolayer.

At step 1475, the fluid in the second conduit is moved past theconstriction into the first conduit and into contact with the first andsecond sensors by air pressure produced by the first pump. The fluid mayinclude a substrate or signal agent and the enzyme remaining within thefirst conduit and immobilized on or near the first and second sensorseither produces an electroactive species from an electroinactivesubstrate or destroys an electroactive substrate. In some embodiments,the fluid may be applied to the first immunosensor and the secondimmunosensor to wash the biological sample from the first secondsensors. A change in current or potential generated by the production ordestruction of the electroactive species at the first and secondsensors, as appropriate to the mode of operation of the sensing device,is recorded as a function of time and determinative of the presence of atarget analyte in the biological sample.

FIG. 15 illustrates a method 1500 of performing an immunoassay fordetermining the concentration of an analyte in a biological sample(e.g., whole blood) in accordance with one embodiment of the invention.At step 1505, a first dry reagent is dissolved into the biologicalsample. The first dry reagent may comprise an enzyme-biomoleculeconjugate (e.g., signal antibodies) configured to bind to the analytesuch as troponin I (TnI) or cardiac troponin I (cTnI). Theenzyme-biomolecule conjugate includes an enzyme conjugated tobiomolecules selected to bind to the analyte of interest. At step 1510,a first complex of the signal antibodies and the analyte is formed in afirst liquid phase comprising the biological sample. At step 1515, asecond dry reagent is dissolved into the biological sample. The seconddry reagent may comprise magnetic beads or microspheres coated withcapture biomolecules (e.g., capture antibodies immobilized on magneticbeads) configured to bind to the analyte. At step 1520, a second complexof the first complex (e.g., signal antibodies bound to the analyte) andthe capture antibodies immobilized on the magnetic beads is formed in asecond liquid phase comprising the biological sample.

At step 1525, the biological sample comprising the first complex and thesecond complex is contacted with a first immunosensor. The firstimmunosensor comprises a capture biomolecule (e.g., latex beads ormicrospheres coated with capture antibodies) immobilized on or near asurface of the first immunosensor. The capture antibodies are configuredto bind to the analyte, to form a third complex localized on or near asolid phase boundary (e.g., a surface) of the first immunosensor. Thethird complex comprises the first complex (e.g., signal antibodies boundto the analyte) and the immobilized capture antibodies of the firstimmunosensor. The localization or capture of the analyte on or near asurface of the first immunosensor is the result of a heterogeneousreaction comprising the formation of the first complex in the firstliquid phase and the formation of the third complex on or near the solidphase boundary. Thus, the first immunosensor may be recognized as aheterogeneous surface capture immunosensor.

At step 1530, the biological sample comprising the first complex and thesecond complex is contacted with a magnetic field localized around asecond immunosensor. The magnetic field is configured to attract themagnetic beads in the biological sample such that the second complex ofthe first complex (e.g., signal antibodies bound to the analyte) and thecapture antibodies immobilized on magnetic beads is localized on or neara surface of the second immunosensor. The localization or capture of theanalyte on or near a surface of the second immunosensor is the result ofa homogenous reaction comprising the formation of the first complex inthe first liquid phase and the formation of the second complex on thesecond liquid phase. Thus, the second immunosensor may be recognized asa homogeneous magnetic bead capture immunosensor.

At step 1535, a fluid (e.g., a wash fluid) may be applied to the firstimmunosensor and the second immunosensor to wash the biological samplefrom the first immunosensor and the second immunosensor. The wash fluidmay comprise a substrate or signal agent (e.g., phosphorylated moleculesuch as 4-aminophenylphosphate). At step 1540, a first signal isdetected and measured at the first immunosensor from a reaction of thesubstrate with the third complex localized on or near the firstimmunosensor. For example a first electrochemical signal is detected andmeasured from the oxidation of an enzymatically produced electroactivespecies (e.g., 4-aminophenol) at a surface of the first immunosensor.The electroactive species is enzymatically produced from the reaction ofthe substrate with the enzyme-biomolecule conjugate in the thirdcomplex. In various embodiments, the substrate is a phosphorylatedmolecule (e.g., 4-aminophenylphosphate) configured such that when aphosphate moiety is removed by the enzyme-biomolecule conjugate (e.g.,one or more antibodies bound to alkaline phosphatase), the moleculebecomes electroactive. At step 1545, a second signal is detected andmeasured at the second immunosensor from a reaction of the substratewith the second complex localized on or near the second immunosensor.For example a second electrochemical signal is detected and measuredfrom the oxidation of an enzymatically produced electroactive species(e.g., 4-aminophenol) at a surface of the second immunosensor. Theelectroactive species is enzymatically produced from the reaction of thesubstrate (e.g., 4-aminophenylphosphate) with the enzyme-biomoleculeconjugate (e.g., one or more antibodies bound to alkaline phosphatase)in the second complex.

At step 1550, a concentration of the analyte in the biological sample isdetermined from at least one of the first signal and the second signal.In some embodiments, the first immunosensor is configured to generatethe first signal as indicative of a concentration of the analyte in afirst range (e.g., an upper concentration range that is greater than alower concentration range) from a reaction of the substrate with thethird complex, while the second immunosensor is configured to generatethe second signal as indicative of a concentration of the analyte in asecond range (e.g., a lower concentration range that is less than anupper concentration range) from a reaction of the substrate with thesecond complex.

In other embodiments in which the analyte is cardiac troponin, the firstimmunosensor is configured to generate the first signal as indicative ofa concentration of the cardiac troponin concentration in a first rangeabove about 1000 pg/mL from a reaction of the substrate with the thirdcomplex, while the second immunosensor is configured to generate thesecond signal as indicative of a concentration of the cardiac troponinconcentration in a second range from about 0 to about 1000 pg/mL from areaction of the substrate with the second complex (where about is +/−10pg/ml around the endpoint of each range). As such, the firstimmunosensor determines the concentration of the cardiac troponin in afirst range above about 1000 pg/mL based on the first signal, and thesecond immunosensor determines the concentration of the cardiac troponinin a second range from about 0 to about 1000 pg/mL based on the secondsignal. In alternative embodiments, the first range is above 2000 pg/mL,the second range is from 0 to 250 pg/ml, and the first signal and thesecond signal in combination (e.g., a weighted average) are indicativeof the concentration of the cardiac troponin concentration in a rangefrom 250 to 2000 pg/ml (where about is +/−10 pg/ml around the endpointof each range). The average may be weighted based on one or more factorsincluding the proximity of the calculated results to defined lower andupper crossover points, the ideality of the shape of the sensor currentversus time plot, and the detection of an error condition at one of thesensors. As such, the first immunosensor determines the concentration ofthe cardiac troponin in a first range above about 2000 pg/mL based onthe first signal, the second immunosensor determines the concentrationof the cardiac troponin in a second range from about 0 to about 250pg/mL based on the second signal, and a combination of the firstimmunosensor and the second immunosensor determines the concentration ofthe cardiac troponin in a third range from about 250 to about 2000 pg/mLbased on the first signal and the second signal.

The lower concentration range (e.g., from about 0 to about 250 pg/mL)may be controlled by a time duration between dissolution of the magneticbeads into the sample and magnetic capture of the magnetic beads on ornear the homogeneous magnetic bead capture immunosensor. In variousembodiments, the time duration is between 1 and 20 minutes, preferablybetween 5 and 10 minutes. The lower concentration range may be furthercontrolled by a dissolved concentration of the magnetic beads in thesample. In some embodiments, the dissolved concentration of the magneticbeads in the sample is in a range from about 10000 to 200000 beads permicroliter, preferably between 10000 to 40000 beads per microliter. Thelower concentration range may be further controlled by an affinity ofeach of the signal antibodies, an avidity of each of the signalantibodies, an affinity of each of the capture antibodies immobilized onthe surface of the magnetic beads, and/or an avidity of each the captureantibodies immobilized on the surface of the magnetic beads. In someembodiments, the affinity of each of the signal antibodies is in a rangefrom about 1×10⁷ to about 1×10¹³ M-1, preferably in a range from about1×10¹⁰ to about 1×10¹³ M-1. In some embodiments, the avidity of each ofthe signal antibodies is in a range from about 1×10⁷ to about 1×10¹³M-1, preferably in a range from about 1×10¹⁰ to about 1×10¹³ M-1. Insome embodiments, the affinity of each of the capture antibodiesimmobilized on the surface of the magnetic beads is in a range fromabout 1×10⁷ to about 1×10¹³ M-1, preferably in a range from about 1×10¹⁰to about 1×10¹³ M-1. In some embodiments, the avidity of each of thecapture antibodies immobilized on the surface of the magnetic beads isin a range from about 1×10⁷ to about 1×10¹³ M-1, preferably in a rangefrom about 1×10¹⁰ to about 1×10¹³ M-1. As should be understood, thelower concentration range (e.g., from about 0 to about 250 pg/mL) may becontrolled by any number of the aforementioned factors alone or incombination, for example, the lower concentration range may becontrolled by at least one of time duration between dissolution of themagnetic beads into the sample and magnetic capture of the magneticbeads on or near the homogeneous magnetic bead capture immunosensor, anaffinity of each of the signal antibodies, an avidity of each of thesignal antibodies, a dissolved concentration of the magnetic beads inthe sample, an affinity of each of the capture antibodies immobilized onthe surface of the magnetic beads, and an avidity of each the captureantibodies immobilized on the surface of the magnetic beads.

The upper concentration range (e.g., above about 2000 pg/mL) may becontrolled by a time duration that the sample is positioned over theheterogeneous surface capture immunosensor. In various embodiments, thetime duration is between 1 and 20 minutes, preferably between 5 and 10minutes. The upper concentration range may be further controlled by anaffinity of each of the signal antibodies, an avidity of each of thesignal antibodies, an affinity of each of the capture antibodiesimmobilized on or near the heterogeneous surface capture immunosensor,and/or an avidity of each the capture antibodies immobilized on or nearthe heterogeneous surface capture immunosensor. In some embodiments, theaffinity of each of the signal antibodies is in a range from about 1×10⁷to about 1×10¹³ M-1, preferably in a range from about 1×10¹⁰ to about1×10¹³ M-1. In some embodiments, the avidity of each of the signalantibodies is in a range from about 1×10⁷ to about 1×10¹³ M-1,preferably in a range from about 1×10¹⁰ to about 1×10¹³ M-1. In someembodiments, the affinity of each of the capture antibodies immobilizedon or near the heterogeneous surface capture immunosensor is in a rangefrom about 1×10¹⁰ to about 1×10¹³ M-1, preferably in a range from about1×10¹⁰ to about 1×10¹³ M-1. In some embodiments, the avidity of each ofthe capture antibodies immobilized on or near the heterogeneous surfacecapture immunosensor is in a range from about 1×10⁷ to about 1×10¹³ M-1,preferably in a range from about 1×10¹⁰ to about 1×10¹³ M-1. As shouldbe understood, the upper concentration range (e.g., above about 2000pg/mL) may be controlled by any number of the aforementioned factorsalone or in combination, for example, the upper concentration range maybe controlled by at least one of time duration that the sample ispositioned over the heterogeneous surface capture immunosensor, anaffinity of each of the signal antibodies, an avidity of each of thesignal antibodies, an affinity of each of the capture antibodiesimmobilized on or near the heterogeneous surface capture immunosensor,and an avidity of each the capture antibodies immobilized on or near theheterogeneous surface capture immunosensor.

FIG. 16 illustrates a method 1600 for determining a concentration of ananalyte in a sample over an extended concentration range in accordancewith one embodiment of the invention. At step 1605, a first signal isdetected and measured at a first immunosensor from a reaction of asubstrate with a third complex localized on or near the firstimmunosensor in accordance with steps 1505-1540 of method 1500. At step1610, a second signal is detected and measured at a second immunosensorfrom a reaction of a substrate with a second complex localized on ornear the second immunosensor in accordance with steps 1505-1545 ofmethod 1500. At step 1615, a first concentration of the analyte in thebiological sample is determined from the first signal and a secondconcentration of the analyte in the biological sample is determined fromthe second signal. At optional step 1620, a weighted average of thefirst concentration and the second concentration is calculated. Theaverage may be weighted based on one or more factors including theproximity of the calculated results to defined lower and upper crossoverpoints, the ideality of the shape of the sensor current versus timeplot, and the detection of an error condition at one of the sensors. Atstep, 1625, the first concentration and the second concentration, oroptionally the weighted average are compared to a predeterminedcrossover concentration point. In various embodiments, the predeterminedcrossover concentration point is 1000 pg/ml, 1200 pg/ml, 1400 pg/ml,1600 pg/ml, 1800 pg/ml, or 2000 pg/ml. At step 1630, when one or both ofthe first concentration and the second concentration, or optionally theweighted average are greater than the predetermined crossoverconcentration point, the first concentration of the analyte determinedfrom the first signal is reported to a user of the device as the finalconcentration of the analyte in the biological sample. At step 1635,when one or both of the first concentration and the secondconcentration, or optionally the weighted average are less than thepredetermined crossover concentration point, the second concentration ofthe analyte determined from the second signal is reported to a user ofthe device as the final concentration of the analyte in the biologicalsample.

FIG. 17 illustrates a method 1700 for determining a concentration of ananalyte in a sample over an extended concentration range in accordancewith one embodiment of the invention. At step 1705, a first signal isdetected and measured at a first immunosensor from a reaction of asubstrate with a third complex localized on or near the firstimmunosensor in accordance with steps 1705-1745 of method 1700. At step1710, a second signal is detected and measured at a second immunosensorfrom a reaction of a substrate with a second complex localized on ornear the second immunosensor in accordance with steps 1705-1750 ofmethod 1700. At step 1715, a first concentration of the analyte in thebiological sample is determined from the first signal and a secondconcentration of the analyte in the biological sample is determined fromthe second signal. At optional step 1720, a weighted average of thefirst concentration and the second concentration is calculated. Theaverage may be weighted based on one or more factors including theproximity of the calculated results to defined lower and upper crossoverpoints, the ideality of the shape of the sensor current versus timeplot, and the detection of an error condition at one of the sensors. Atstep, 1725, the first concentration and the second concentration, oroptionally the weighted average are compared to a predeterminedcrossover concentration zone. In various embodiments, the predeterminedcrossover concentration zone is 400 to 2000 pg/ml, 600 to 1800 pg/ml,400 to 1800 pg/ml, 800 to 1600 pg/ml, or 250 to 2000 pg/mL.

At step 1730, when one or both of the first concentration and the secondconcentration, or optionally the weighted average are greater than thepredetermined crossover concentration zone, the first concentration ofthe analyte determined from the first signal is reported to a user ofthe device as the final concentration of the analyte in the biologicalsample. At step 1735, when one or both of the first concentration andthe second concentration, or optionally the weighted average are lessthan the predetermined crossover concentration zone, the secondconcentration of the analyte determined from the second signal isreported to a user of the device as the final concentration of theanalyte in the biological sample. At step 1740, when both of the firstconcentration and the second concentration, or optionally the weightedaverage are within the predetermined crossover concentration zone, theweighted average of the first concentration and the second concentrationis reported to a user of the device as the final concentration of theanalyte in the biological sample.

FIG. 18 shows a graph 1800 that illustrates the impact of being able todetermine a concentration of an analyte in a sample over an extendedconcentration range in accordance with various embodiments of theinvention. A microfabricated extended range sensor chip may include thefirst immunosensor 1805 (e.g., a low-end sensitivity amperometric sensorwith an immobilized layer of capture antibodies) and the secondimmunosensor 1810 (e.g., a high-end sensitivity amperometric sensor witha magnetic field to attract magnetic beads with an immobilized layer ofcapture antibodies), as described herein. Graph 1800 shows that thefirst immunosensor 1805 is particularly well suited for detectinganalytes having a higher concentration, for example, greater than 400pg/ml, while the second immunosensor 1810 is particularly well suitedfor detecting analytes having a lower concentration, for example, lessthan 2000 pg/ml. Accordingly, by using a system having a sensor chip asdescribed herein with both the first immunosensor 1805 (e.g., a low-endsensitivity amperometric sensor with an immobilized layer of captureantibodies) and the second immunosensor 1810 (e.g., a high-endsensitivity amperometric sensor with a magnetic field to attractmagnetic beads with an immobilized layer of capture antibodies), it ispossible to extend the range of concentrations that an analyte may bedetected at by using the first and second signals generated at therespective immunosensors as described above with respect to methods1500, 1600, and 1700.

While the invention has been described in terms of various preferredembodiments, those skilled in the art will recognize that variousmodifications, substitutions, omissions and changes can be made withoutdeparting from the spirit of the present invention. It is intended thatthe scope of the present invention be limited solely by the scope of thefollowing claims. In addition, it should be appreciated by those skilledin the art that a plurality of the various embodiments of the invention,as described above, may be coupled with one another and incorporatedinto a single reader device.

We claim:
 1. An immunoassay system for determining a concentration of ananalyte in a sample over an extended concentration range, the systemcomprising: a first electrochemical sensor including an immobilizedlayer of antibodies configured to bind to a first complex of signalantibodies and the analyte to form a second complex; a secondelectrochemical sensor having a magnetic field disposed locally aroundthe second electrochemical sensor, wherein the magnetic field isconfigured to attract magnetic beads onto a surface of the secondelectrochemical sensor such that a third complex of the first complexand antibodies immobilized on the magnetic beads is localized on thesecond immunosensor; a fluid comprising a substrate; one or moreprocessors; and non-transitory machine readable storage medium storing aset of instructions which when executed by the one or more processorscause the one or more processors to: measure a first electrochemicalsignal at the first electrochemical sensor from a reaction of thesubstrate with the second complex; measure a second electrochemicalsignal at the second electrochemical sensor from a reaction of thesubstrate with the third complex; determine a first concentration of theanalyte in the sample from the first electrochemical signal; determine asecond concentration of the analyte in the sample from the secondelectrochemical signal; determine a weighted average of the firstconcentration and the second concentration; compare the weighted averageof the first concentration and the second concentration to apredetermined crossover concentration point; when the weighted averageis above the crossover concentration point, report the firstconcentration as a final concentration of the analyte in the sample fromthe first electrochemical signal; and when the weighted average is belowthe crossover concentration point, report the second concentration asthe final concentration of the analyte in the biological sample.
 2. Thesystem of claim 1, further comprising a first reagent region coated withthe signal antibodies for the analyte.
 3. The system of claim 2, furthercomprising a second reagent region coated with the magnetic beads. 4.The system of claim 4, wherein first electrochemical sensor, secondelectrochemical sensor, the first reagent region, and the second reagentregion are located on a sensor chip.
 5. The system of claim 1, furthercomprising a composite material including a binder and a particulatemagnetic material, the particulate magnetic material configured togenerate the magnetic field.
 6. The system of claim 5, wherein thebinder is a polyimide or polyvinyl alcohol (PVA).
 7. The system of claim5, wherein the particulate magnetic material is comprised of neodymiumiron boron (NdFeB) alloy or aluminum nickel cobalt (AlNiCo) alloy. 8.The system of claim 1, wherein the signal antibodies are conjugated withan enzyme.
 9. The system of claim 8, wherein the enzyme is alkalinephosphatase.
 10. The system of claim 9, wherein the substrate is aphosphorylated molecule configured such that when a phosphate moiety isremoved the molecule becomes electroactive.
 11. The system of claim 1,wherein the analyte is cardiac troponin I (cTnI).
 12. An immunoassaysystem for determining a concentration of an analyte in a sample over anextended concentration range, the system comprising: a firstelectrochemical sensor including an immobilized layer of antibodiesconfigured to bind to a first complex of signal antibodies and theanalyte to form a second complex; a second electrochemical sensor havinga magnetic field disposed locally around the second electrochemicalsensor, wherein the magnetic field is configured to attract magneticbeads onto a surface of the second electrochemical sensor such that athird complex of the first complex and antibodies immobilized on themagnetic beads is localized on the second immunosensor; a fluidcomprising a substrate; one or more processors; and non-transitorymachine readable storage medium storing a set of instructions which whenexecuted by the one or more processors cause the one or more processorsto: measure a first electrochemical signal at the first electrochemicalsensor from a reaction of the substrate with the second complex; measurea second electrochemical signal at the second electrochemical sensorfrom a reaction of the substrate with the third complex; determine afirst concentration of the analyte in the sample from the firstelectrochemical signal; determine a second concentration of the analytein the sample from the second electrochemical signal; determine aweighted average of the first concentration and the secondconcentration; compare the weighted average of the first concentrationand the second concentration to a predetermined crossover concentrationzone; when the weighted average is above the crossover concentrationzone, report the first concentration as a final concentration of theanalyte in the sample from the first electrochemical signal; when theweighted average is below the crossover concentration zone, report thesecond concentration as the final concentration of the analyte in thebiological sample from the second electrochemical signal; and when theweighted average is within the crossover concentration zone, report theweighted average as the final concentration of the analyte in thebiological sample.
 13. The system of claim 12, further comprising afirst reagent region coated with the signal antibodies for the analyte,and a second reagent region coated with the magnetic beads.
 14. Thesystem of claim 13, wherein first electrochemical sensor, secondelectrochemical sensor, the first reagent region, and the second reagentregion are located on a sensor chip.
 15. The system of claim 12, furthercomprising a composite material including a binder and a particulatemagnetic material, the particulate magnetic material configured togenerate the magnetic field.
 16. The system of claim 15, wherein thebinder is a polyimide or polyvinyl alcohol (PVA).
 17. The system ofclaim 15, wherein the particulate magnetic material is comprised ofneodymium iron boron (NdFeB) alloy or aluminum nickel cobalt (AlNiCo)alloy.
 18. The system of claim 12, wherein the analyte is cardiactroponin I (cTnI).
 19. A non-transitory machine readable storage mediumhaving instructions stored thereon that when executed by one or moreprocessors cause the one or more processors to perform a methodcomprising: measuring a first signal at a first immunosensor from areaction of a signal agent with a first complex of signal antibodies,analyte, and capture antibodies immobilized on a surface of the firstimmunosensor; measuring a second signal at a second immunosensor from areaction of the signal agent with a second complex of the signalantibodies, the analyte, and capture antibodies immobilized on magneticbeads that are localized on or near a surface of the second immunosensorvia a magnetic field; determining a first concentration of the analytein the sample from the first electrochemical signal; determining asecond concentration of the analyte in the sample from the secondelectrochemical signal; determining a weighted average of the firstconcentration and the second concentration; comparing the weightedaverage of the first concentration and the second concentration to apredetermined crossover concentration point; when the weighted averageis greater than the crossover concentration point, reporting the firstconcentration as a final concentration of the analyte in the sample fromthe first signal; and when the weighted average is less than thecrossover concentration point, reporting the second concentration as thefinal concentration of the analyte in the biological sample from thesecond signal.
 20. A non-transitory machine readable storage mediumhaving instructions stored thereon that when executed by one or moreprocessors cause the one or more processors to perform a methodcomprising: measuring a first signal at a first immunosensor from areaction of a signal agent with a first complex of signal antibodies,analyte, and capture antibodies immobilized on a surface of the firstimmunosensor; measuring a second signal at a second immunosensor from areaction of the signal agent with a second complex of the signalantibodies, the analyte, and capture antibodies immobilized on magneticbeads that are localized on or near a surface of the second immunosensorvia a magnetic field; determining a first concentration of the analytein the sample from the first electrochemical signal; determining asecond concentration of the analyte in the sample from the secondelectrochemical signal; determining a weighted average of the firstconcentration and the second concentration; comparing the weightedaverage of the first concentration and the second concentration to apredetermined crossover concentration zone; when the weighted average isgreater than the crossover concentration zone, reporting the firstconcentration as a final concentration of the analyte in the sample fromthe first signal; when the weighted average is less than the crossoverconcentration zone, reporting the second concentration as the finalconcentration of the analyte in the biological sample from the secondsignal; and when the weighted average is within the crossoverconcentration zone, reporting the weighted average as the finalconcentration of the analyte in the biological sample from the firstsignal and the second signal.